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CN101245101B - Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146 - Google Patents

Antihuman CD146 monoclone antibody, composition containing the same, and method for testing dissolubility CD146 Download PDF

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CN101245101B
CN101245101B CN 200810057260 CN200810057260A CN101245101B CN 101245101 B CN101245101 B CN 101245101B CN 200810057260 CN200810057260 CN 200810057260 CN 200810057260 A CN200810057260 A CN 200810057260A CN 101245101 B CN101245101 B CN 101245101B
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CN101245101A (en
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阎锡蕴
张莹
郑超固
杨东玲
冯静
卢迪
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Institute of Biophysics of CAS
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Abstract

The invention relates to a group of anti-human CD146 molecules which are developed by utilizing the biological technology and an established high-sensitive sandwich ELISA method for detecting the solubility CD146. The invention includes anti-human CD146 mouse monoclonal antibodies of AA1, AA2, AA3, AA4, AA5 and AA7, and the method of utilizing the combination of the antibodies and the sandwich ELISA specificity for detecting the solubility CD146. The group of antibodies can identify the human source CD146 on the molecular, the cellular and the tissue levels, which can be divided into two categories based on the identified different epitopes. The sandwich ELISA method which is combined by the antibody AA1 and another strain of anti-human CD146 mouse monoclonal antibody AA98 can detect the solubility CD146 at each milliliter nano-gram level. The antibodies and the detection means can become the effective detection or diagnosis tools and method in the basic research or the clinical application and provide good carriers for the targeted therapy of the CD146-related diseases at the same time.

Description

The monoclonal antibody of anti-human CD146 comprises its composition, detects the method for sMe1-CAM
Technical field
The invention belongs to molecular biology and biological technical field.Specifically the present invention relates to the mouse monoclonal antibody of one group of anti-human CD146, this group antibody can and be organized horizontal specific recognition people source CD146 albumen at biochemistry, cell.The invention still further relates to the method that a kind of sandwich ELISA detects sMe1-CAM, capture antibody is the mouse monoclonal antibody that relates among the present invention in this detection method, and detecting antibody is the mouse monoclonal antibody AA98 that relates to among the biotin labeled patent of invention ZL 991075862.
Background technology
CD146 has another name called MUC18, and Mel-CAM/MCAM is a kind of cell adhesion molecule of contactin.CD146 born of the same parents have five immunoglobulin like domain (V-V-C2-C2-C2) (HolnessandSimmons 1994) outward, and by high glycosylation, the iuntercellular of the non-dependence of mediation calcium ion sticks mutually.
CD146 finds it is melanomatous marker molecule the earliest, participates in melanomatous transfer and deterioration (Lehmann, Riethmuller et al.1989; Sers, Kirsch et al.1993).The overexpression of CD146 in the melanochrome tumour strengthened sticking between the tumour cell, and for the invasion and attack of tumour with shift extremely important (Johnson, Rothbacher et al.1993).Studies show that, the expression of CD146 is directly related with the transfer ability of melanoma cell, and overexpression CD146 can significantly strengthen invasion and attack and transfer ability (Bani, the Rak etal.1996 of tumour cell in the melanoma cell of not expressing CD146; Shih, Speicher et al.1997; Xie, Luca et al.1997).In addition, CD146 also expresses in a small amount of healthy tissues, such as unstriated muscle, (Shih 1999) such as blood vessel endothelium and trophoderms.
Simultaneously, CD146 also is widely regarded as specificity marker molecule (Bardin, the Frances et al.1996 of vascular endothelial cell; Bardin, Anfosso et al.2001).In research before, anti-human CD146 antibody is employed circulating endothelial cells (George, the Poncelet et al.1991 that detects in the blood; Bardin, George et al.1996; Solovey, Gui et al.2001).Because this feature, in some disease with endothelial injury, obscission, CD146 can be used as the reference index of disease process.Other there are some researches show that the CD146 molecule also is expressed in monocyte, has on the T lymphocyte of the activation of being reported in high expression level (Pickl, Majdic et al.1997) is also arranged.As other a lot of adhesion molecules, the CD146 molecule exists (Neidhart, Wehrli et al.1999 with cytolemma and soluble form (soluble CD146, sCD146) in the body; Bardin, Moal et al.2003).Studies show that the expression level of sCD146 in the serum of rheumatic arthritis synovia and chronic renal failure patients is apparently higher than the normal people.
Anti-human CD146 antibody has embodied the result for the treatment of of very strong inhibition tumor growth in experimentation on animals.The anti-CD146 human antibody of Mills etc. exploitation, ABX-MA1 significantly growth of check melanin tumour in nude mice model, invasion and attack and to the transfer (Mills, Tellez et al.2002) of lung.Described in the patent of invention ZL 991075862, the anti-CD146 mouse monoclonal antibody AA98 of exploitation has the effect that suppresses tumor-blood-vessel growth, and the obviously growth (Yan, Lin et al.2003) of inhibition tumour (such as liver cancer, carcinoma of the pancreas etc.) in multiple nude mice lotus knurl experiment.The mechanism that further research has also disclosed the inhibition tumor-blood-vessel growth of AA98 realizes (Bu, Gao et al.2006) by suppressing MAPK phosphorylation and NF kB activation.
Although organizing different blood vessel endotheliums, the more existing reports of the antibody of anti-human source CD146, these antibody but have different combinations active.For example S-endol can in conjunction with the endotheliocyte of various types of blood vessels, comprise (George, Poncelet et al.1991) such as artery, arteriole, vein, Venule, capillary vessel, high endothelium venule and lymph vessels systems.And the anti-CD146 antibody of another strain MUC18 is only in conjunction with capillary vessel and high endothelium venule (Kuzu, Bicknell et al.1993).S-endol MUC18 debond (Bardin, Frances et al.1996) in conjunction with the umbilical vein endothelial tissue.In addition, P1H12 can be simultaneously in conjunction with the vascular endothelial cell (StCroix, Rago et al.2000) of tumour and healthy tissues, and the specific vascular endothelial cell in conjunction with tumor tissues of AA98 (Yan, Lin et al.2003).These evidences show, probably different antibodies epi-positions is in different tissues and the exposure in microenvironment difference to some extent.Therefore, for different epi-position exploitation antibody on the CD146 albumen, and study it for the binding ability of different tissues, the function of CD146 on vascular endothelial cell is very necessary for illustrating.
Summary of the invention
The present invention utilizes the CD146 protein immunization mouse of natural purifying, obtains hybridoma.Method with ELISA is screened the antibody that strong binding ability is arranged with recombinant expressed CD146 protein extracellular, obtain six strain antibodies, difference called after AA1, AA2, AA3, AA4, AA5 and AA7, obtain simultaneously the hybridoma of these antibody of secretion, be successively 8E8,8F4, A5, G10, H5 1. with H5 2..This six strain antibody all belongs to IgG1, the κ hypotype.The experiment confirm of ELISA and immunoblotting the specific binding of these antibody and CD146 molecule.
Utilize the outer different structural domain of recombinant expressed CD146 albumen born of the same parents and the method for immunoblotting, confirm that AA1 and AA2 identification epi-position (sequence is the position 24-128 in the people CD146 sequence of SEQ ID NO:1) is positioned at first IgV structural domain, and AA3, AA4, AA5 and AA7 identification epi-position (sequence is the position 335-400 in the people CD146 sequence of SEQ ID NO:1) are positioned at second IgC2 structural domain.Therefore, this six strain antibody is divided into two classes according to different epitopes: V1 class (comprising AA1 and AA2) and C2-2 class (comprising AA3, AA4, AA5 and AA7).
Cellular immunofluorescence, immunoprecipitation and immunohistochemical experiment discovery V1 antibody-like can and organize level to identify simultaneously the CD146 protein of native conformation and sex change at molecule, cell, and the C2-2 antibody-like is only identified the CD146 protein of sex change.This has confirmed that the exposure of different epitopes under different situations is different.
The present invention utilizes the V1 antibody-like simultaneously, such as the mouse monoclonal antibody AA98 that relates among AA1 and the biotin labeled patent of invention ZL991075862, has developed the method for the sandwich ELISA of a cover, for detection of sMe1-CAM molecule in solution and the body fluid.Commercialization detection method report (the CyQuant ELISA assay kit that provides before comparing, Bioxytex, Marseille, France) double antibodies sandwich ELISA (sensitivity is 10ng/ml), sandwich ELISA method sensitivity of the present invention has improved an order of magnitude, reaches 1ng/ml.This method can be used in the detection of sMe1-CAM in human blood and the cerebrospinal fluid, and has clinical diagnostic applications value in the illness of CD146 abnormal expression.Use the method among the present invention, find that the CD146 molecule content among the patients serum significantly raises, and has directive significance and using value for clinical diagnosis in the autoimmune diseases such as systemic vasculitis.
Particularly, the present invention relates to a kind of people CD146 epitope, its aminoacid sequence is shown in SEQ IDNO:24 or the SEQ ID NO:25.
In one embodiment, the present invention relates to a kind of anti-human CD146 antibody, it is characterized in that it can the above-mentioned people CD146 of specific recognition epitope.
Preferably, described anti-human CD146 antibody is characterised in that and comprises heavy chain of antibody and light chain of antibody, wherein said heavy chain of antibody comprises the CDR1 that the aminoacid sequence by SEQ ID NO:26 as CDR forms, the CDR2 that is formed by the aminoacid sequence of SEQ ID NO:27 and formed by the CDR3 that the aminoacid sequence of SEQID NO:28 forms
Described light chain of antibody comprises the CDR1 that the aminoacid sequence by SEQ ID NO:29 as CDR forms, the CDR2 that is comprised of the aminoacid sequence of SEQ ID NO:30 and be comprised of the CDR3 that the aminoacid sequence of SEQ IDNO:31 forms.
More preferably, the heavy chain of described antibody is comprised of the aminoacid sequence of SEQ ID NO:32, and light chain is comprised of the aminoacid sequence of SEQ ID NO:33.
In another embodiment, the present invention relates to a kind of anti-human CD146 antibody, be characterised in that to comprise heavy chain of antibody and light chain of antibody,
Wherein said heavy chain of antibody comprises the CDR1 that the aminoacid sequence by SEQ ID NO:34 as CDR forms, the CDR2 that is formed by the aminoacid sequence of SEQ ID NO:35 and formed by the CDR3 that the aminoacid sequence of SEQID NO:36 forms,
Described light chain of antibody comprises the CDR1 that the aminoacid sequence by SEQ ID NO:37 as CDR forms, the CDR2 that is comprised of the aminoacid sequence of SEQ ID NO:38 and be comprised of the CDR3 that the aminoacid sequence of SEQ IDNO:39 forms.
Preferably, the heavy chain of described antibody is comprised of the aminoacid sequence of SEQ ID NO:40, and light chain is comprised of the aminoacid sequence of SEQ ID NO:41.
In another aspect of this invention, the antibody that also relates to above-mentioned anti-human CD146 is for the preparation of the application in the pharmaceutical composition for the treatment of tumour.Preferably, described tumour is melanoma, liver cancer, carcinoma of the pancreas.
In another aspect of the present invention, the antibody that also relates to above-mentioned anti-human CD146 is for the preparation of the application in the diagnostic reagent of the imaging level diagnosis of the human tumor of target CD146.
In yet another aspect, the invention still further relates to a kind of composition, for example be used for the treatment of the pharmaceutical composition of tumour, it comprises the anti-human CD146 antibody of above-mentioned either side, and pharmaceutical carrier.
When being used for this paper, " pharmaceutical carrier " comprise any He all solvents, dispersion medium, Drug coating, antibacterial agent and anti-mycotic agent that physiology is fit to, etc. blend absorption and delay reagent etc.Preferably, described carrier is suitable for the using of intravenous, intramuscular, subcutaneous, parenteral, backbone or epidermis (for example, by injection or inculcate).
Can use composition of the present invention by many methods known in the art.As it will be understood by those skilled in the art that using path and/or pattern will depend on results needed and change.
Use the compounds of this invention in order to use the path by some, be coated with compound with a kind of material, or with the common administered compound of a kind of material may be essential to prevent its inactivation.For example, can be with suitable carrier, for example liposome or thinner come to use described compound to the experimenter.Medicinal diluent comprises salt solution and aqueous buffer.
Pharmaceutical carrier comprises for the Injectable solution of the standby sterilization of immediate system or sterile water solution or dispersion and the sterile powder of dispersion.These media and reagent are known in the art for being applied as of active medicinal matter.
When being used for this paper, phrase " parenteral using " and " through parenteral administration " expression except intestines in topical application the common mode of administration of injecting passed through, including, but not limited to, under in intravenous, intramuscular, endarterial, the sheath, in the capsule, the socket of the eye, intracardiac, intracutaneous, endoperitoneal, transtracheal, subcutaneous, subepidermal, IA, the capsule, subarachnoid, intravertebral, peridural, intrasternal injection and inculcating.
Which kind of no matter selects use the path, by being ordinary method well known by persons skilled in the art, can be mixed with pharmaceutically acceptable formulation with the compound of the present invention that suitable hydrated form and/or pharmaceutical compositions of the present invention are used.
For particular patient, composition and method of application, the actual dose level of the activeconstituents of pharmaceutical composition of the present invention can change the therapeutic response that obtains effectively to realize ideal and can be to the amount of the virose activeconstituents of patient's tool.Selected dosage level will depend on many pharmacokinetics factors, it comprises the activity of used particular composition of the present invention, the path of using, the time of using, the excretion rate of used specific compound, the time length for the treatment of, the other medicines, compound and/or the material that are combined with used specific compound, patient's to be treated age, sex, body weight, disease condition, general health and former history of disease and the well-known similar factor of medical field.
Described composition must be aseptic and fluid, the degree that can transmit by syringe to arrive composition.Except water, carrier is the isotonic buffer salts solution preferably.
In yet another aspect, the invention still further relates to the hybridoma cell strain of the anti-human CD146 antibody of secretion, preserving number is respectively CGMCC NO:2310 and CGMCC NO:2311.
In yet another aspect, the invention still further relates to the nucleic acid of the polypeptide that coding can assemble with each the other antibody chain that defines below, wherein said polypeptide is any of following polypeptide
A) heavy chain of antibody, it is one of above-mentioned defined heavy chain of antibody;
B) light chain of antibody, it is one of above-mentioned defined light chain of antibody.
Simultaneously, the invention still further relates to the expression vector that comprises according to the nucleic acid of claim 11, it can express described nucleic acid in protokaryon or eukaryotic host cell.And the protokaryon or the eukaryotic host cell that comprise described expression vector.
In another aspect of the present invention, also relate to a kind of method for preparing in conjunction with the antibody of people CD146, it is characterized in that in protokaryon or eukaryotic host cell, expressing the nucleic acid of above-mentioned encoding antibody heavy chain and the nucleic acid of encoding antibody light chain, and from described cell, reclaim described polypeptide.
In another aspect of the present invention, relate to the method for a kind of people of detection CD146, it utilizes above-mentioned anti-human CD146 antibody, is undertaken by enzyme-linked immunosorbent assay.Preferably, described enzyme-linked immunosorbent assay is sandwich enzyme-linked immunoabsorption, wherein with one or more of above-mentioned anti-human CD146 antibody as capture antibody, with the mouse monoclonal antibody AA98 among the Chinese patent ZL 991075862 as detecting antibody.More preferably, described people CD146 is present in the body fluid.Even more preferably, described body fluid is tissue juice, serum, lymph liquid or cerebrospinal fluid.Described body fluid can pick up from the autoimmune disorder patients such as systemic vasculitis, systemic lupus erythematous, multiple sclerosis and Guillain Barre syndrome or/and the patient of the illnesss such as chronic kidney hypofunction.
In another aspect of the present invention, the derivative that also relates to the antibody of described anti-human CD146, wherein said derivative be described antibody and biomarker (such as nano materials such as the conventional fluorescence dye such as vitamin H, HRP, alkaline phosphatase, FITC, PE, Cy3, Cy5, nanometer magnetic beads), antitumor drug (carboplatin as be known in the art, cis-platinum, 5-Fluorouracil etc.), toxin (such as Ricin, lymphotoxin etc.), radioactivity agent (as 131Iodine, 90Yttrium, radioactivity copper etc.) product of combination.
Those of ordinary skill in the art in conjunction with the knowledge of prior art, can obtain said derivative based on the instruction of specification sheets about anti-human CD146 antibody fully without difficulty, and then the effect of definite derivative.
Innovative point of the present invention is: (1) has developed one group of mouse monoclonal antibody for people CD146 albumen; (2) disclosed this and organized the epitope of anti-CD146 antibody recognition, and confirmed to identify the antibody of different epi-positions, at molecule, cell and organize on the level of the marked difference that is combined with to(for) CD146 albumen; (3) developed the detection that a kind of highly sensitive sandwich ELISA method is used for the sMe1-CAM molecule.
Description of drawings
Fig. 1: AA1-AA7 specific recognition recombination human source CD146 molecule extracellular region section albumen.
Fig. 2: the outer recombinant protein schematic diagram of various CD146 born of the same parents.
The epitope of Fig. 3: AA1-AA5 and AA7 is identified.Upper figure is the protein electrophoresis figure of D1-D5.Figure below is to utilize respectively AA1-AA5 and AA7 to carry out the binding ability that immunoblot experiment detects itself and D1-D5.
Fig. 4: AA1-AA5 and AA7 identify reduction and non-reduced CD146 albumen in immunoblotting.Swimming lane 1-6 is non-reducing A375 cell pyrolysis liquid, and swimming lane 7-12 is the A375 cell pyrolysis liquid of reduction.
Fig. 5: AA1-AA5 and AA7 are used for the CD146 Molecular Detection of flow cytometry.MIgG is as negative control among the figure.Only has the CD146 molecule in AA1 and the AA2 identification viable cell.
Fig. 6: AA1-AA5 and AA7 are used for the cellular immunofluorescence experiment and detect CD146 on the cytolemma.AA98 is as the positive control of experiment, and mIgG is as the negative control of experiment.Only have AA1 and AA2 and the AA98 can be in conjunction with CD146 on the cytolemma, arrow only shows one of them.
Fig. 7: AA1-AA5 and AA7 are used for immunoprecipitation experiment and catch the CD146 of cell pyrolysis liquid.Swimming lane 1-7 uses respectively mIgG, and AA1-5 and AA7 carry out the albumen composition that immunoprecipitation is caught, and swimming lane 8 is lysis supernatants, as positive control, carries out immunoblotting with AA98 and detects.
Fig. 8: AA1-AA5 and AA7 carry out the frozen section immunohistochemical experiment and detect the CD146 molecule of organizing level.AA98 is as the positive control of experiment, and CD31 is used for label vascular endotheliocyte (as shown by arrows).As shown by arrows, only have AA1 and the AA2 can be in conjunction with the CD146 molecule of organizing level on the frozen section.
Fig. 9: sandwich ELISA detects the typical curve of 5ng/ml-320ng/ml standard model.
Figure 10: sandwich ELISA detects the typical curve of 5ng/ml-80ng/ml standard model, and fit equation.
Figure 11: the collection of illustrative plates of HMV6g carrier, it contains the MBP-His label.
Hybridoma cell strain 8E8 and G10 (it is secretory antibody AA1 and AA4 respectively) are preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on January 24th, 2008, China, Beijing, No. 13, one in Zhong Guan-cun, Haidian District north), preserving number is respectively CGMCC No.2310 and CGMCC No.2311.
Embodiment
Describe the present invention in detail below by embodiment.Persons of ordinary skill in the art may appreciate that following embodiment only is presented for purposes of illustration.The spirit and scope of the present invention are limited by accompanying claim.
Embodiment 1: the preparation of monoclonal antibody AA1-AA5 and AA7 and evaluation.
(KohlerandMilstein 1975 to use hybridoma technology; Yeh, Hellstrom et al.1979; Yeh, Hellstrom et al.1982) produce and screening acquisition antibody A A1-AA6 and AA7.Be summarized as follows: (its aminoacid sequence is shown in SEQID NO:1 from separating natural CD146 albumen human umbilical vein's endotheliocyte, nucleotide sequence is shown in SEQ ID NO:2), according to (Yan, Lin et al.2003) the monoclonal antibody AA98 antigen purification method purifying of describing, it is carried out immunization as immunogen to BALB/C mice (Beijing Experimental Animal Center), each peritoneal injection 100 μ g albumen/mouse, every fortnight once, totally three times.Before the extracting spleen cell booster immunization once, peritoneal injection 100 μ g albumen/mouse.Booster immunization three days is afterwards got spleen, and splenocyte is suspended in the RPMI substratum.In the presence of polyoxyethylene glycol (PEG), splenocyte and SP2/0-Ag14 rat bone marrow tumour cell (ATCC) are merged, and with the HAT selective medium hybridoma is screened.
The method screening of utilization Enzyme-linked Immunosorbent Assay (ELISA) can produce the hybridoma cell clone of the anti-human CD146 antibody of high-affinity in a large number.Use recombinant expressed CD146 molecule extracellular region protein, rhCD146 (sequence is the position amino acid 24-552 in the people CD146 sequence of SEQ ID NO:1) as envelope antigen, detects the Hybridoma Cell Culture supernatant.Concrete way is that at first spending the night at elisa plate is coated with 50 μ l, 1 μ g/ml rhCD146 albumen, washes three times with PBS.Add 2% bovine serum albumin (Ameresco) sealing 1 hour.Then the hybridoma culture supernatant, enzyme labelled antibody (the sheep anti mouse HRP of dilution in 1: 5000, Santa Cruz) and substrate (200ng/ml TMB (Ameresco), the 0.03%H that successively add secretory antibody 2O 2(Beijing chemical reagents corporation) pH4.5), carries out the ELISA screening.Hybridoma cell strain 8E8,8F4, A5, G10, the H5 that has obtained six secretion high-affinity antibodies by three-wheel screening 1. with H5 2., the antibody of its secretion is corresponding called after AA1, AA2, AA3, AA4, AA5 and AA7 respectively.Hybridoma cell strain 8E8 and G10 (it is secretory antibody AA1 and AA4 respectively) are preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on January 24th, 2008, China, Beijing, No. 13, one in Zhong Guan-cun, Haidian District north), preserving number is respectively CGMCC No.2310 and CGMCC No.2311.And hybridoma cell strain AA6 is the ELISA negative hybridoma clone in when screening, and is very weak with the CD146 binding ability.
Respectively a large amount of cultivate amplified hybridization oncocyte 8E8,8F4, A5, G10, H5 1. with H5 2., collect respectively the culture supernatant that contains AA1, AA2, AA3, AA4, AA5 and AA7 antibody and be used for antibody function and identify.Respectively 2. 1. hybridoma 8E8,8F4, A5, G10, H5 are made cell suspension with serum-free RPMI-1640 substratum with H5 in addition, for the preparation of antibody ascites.The preparation method of ascites is summarized as follows: six week BALB/C mice abdominal injection pristane in age (Sigma) 0.5ml/ only.After 10 days, with hybridoma suspension 1 * 10 6Individual/ml is inoculated in the BALB/C mice abdominal cavity, and 0.5ml/ only approximately after ten days, collects ascites, the centrifuging and taking supernatant.
By protein A affinity chromatography, monoclonal antibody purification AA1, AA2, AA3, AA4, AA5 and AA7 from culture supernatant or ascites.With monoclonal antibody purification sterile filtration, and refrigeration or freezing preservation.
Use BD Pharmingen company's murine antibody hypotype identification kit (catalog number (Cat.No.) 550487), with AA98 described in the patent of invention ZL 991075862 (lower with) as testing positive control, the ELISA method identifies antibody A A1, AA2, AA3, AA4, AA5 and AA7 and all belongs to the IgG1 hypotype, and is as shown in table 1.
Table 1 antibody A A1, AA2, AA3, AA4, AA5, AA7 and AA98 antibody typing
Antibody The heavy chain hypotype Light chain subtype
AA1 IgG1 κ
AA2 IgG1 κ
AA3 IgG1 κ
AA4 IgG1 κ
AA5 IgG1 κ
AA7 IgG1 κ
AA98 IgG2a κ
The ELISA method is for the identification of the AA1-AA5 of purifying and six kinds of antibody of AA7 binding specificity for CD146 albumen.Alkaline phosphatase (the Alkaline Phosphatase of the His-rhCD146 that the 6xHis-tag of prokaryotic expression merges (amino acid that by 6xHis-tag and sequence is the position 24-552 in the people CD146 sequence of SEQ ID NO:1 forms) and eukaryotic expression, be called for short AP, its sequence is as shown in SEQ ID NO:3) sCD146-AP that merges (being that the bonding pad that the amino acid of the position 24-559 in the people CD146 sequence of SEQ ID NO:1 and AP that sequence is SEQ ID NO:3 and sequence are SEQ ID NO:42 forms by sequence) is used as respectively envelope antigen (their structural representation is referring to Fig. 2) in experiment.
Wherein, the His-rhCD146 preparation method briefly introduces as follows: the Nucleotide of position 70-1656 in the cDNA sequence (SEQ ID NO:2) of people CD146 is cloned on pET28b (Novagen) carrier, utilizes restriction enzyme NdeI and NotI (NEB provides) and primer 5 '-AA C ATA TGG TGC CCG GAG AGG CTG AGC AG-3 ' (SEQ ID NO:4) and 5 '-AAGCGG CCG CCA GCT TTC TCT CTG TGG AG-3 ' (SEQ ID NO:5).In E.ColiBL21 DE3 (Invitrogen) bacterial strain, express, exist with the occlusion body form, obtain required antigen after washing and the renaturation.Protein expression and inclusion body washing methods are seen following D1, D2 and D4 protein expression and inclusion body washing methods.
The renaturing inclusion bodies step is as follows:
1. transfer concentration to 1mg/ml the inclusion body protein of dissolving, common 1ml packs in the dialysis tubing, outer liquid is with 140ml 6M urea element, the 200mM arginine, 25mM Tris (pH8.0), 150mM NaCl, 2mM reduced glutathion (GSH), 4 ℃ of 1mM Sleep-promoting factor B (GSSG) leave standstill dialysed overnight.
2. pour out above-mentioned 50ml extracellular fluid dialysis, fill into 50ml diluent [600mM arginine, 25mM Tris (pH8.0), 150mM NaCl, 2mM GSH, 1mM GSSG.].This moment outer liquid urea concentration be 4M.Dialysed 6 hours for 4 ℃.
3. pour out the 75ml extracellular fluid dialysis, fill into the 75ml diluent, the urea final concentration is 2M.Dialysed 6 hours for 4 ℃.
4. join 200ml 400mM arginine, 25mM Tris, 150mM NaCl, 2mM GSH, 1mM GSSG, dialysed overnight.Urea concentration is 0M, and arginine concentration is 400mM.
5. pour out the 100ml extracellular fluid dialysis, fill into 100ml 25mM Tris, 150mM NaCl.Whole solution is the 200mM arginine, 25mM Tris, 150mM NaCl, 1mM GSH, 0.5mMGSSG.Dialysed 6 hours for 4 ℃.
6. repeating step 5.
7. with 900ml 25mM Tris 150mM NaCl dialysed overnight.
8. renew bright 1L 25mM Tris 150mM NaCl dialysis 6 hours.
9. mensuration protein content, the SDS-PAGE method detects.Concentration can reach more than the 500 μ g/ml.Can be further concentrated by ultrafiltration or Ni-NTA affinity chromatography.Dialysis provides by Sigma company with chemical reagent.)
The preparation process of sCD146-AP is simply as follows: the coding nucleotide sequence of fusion rotein (SEQID NO:6) is cloned on the pCMV-SPORTS6 carrier (OpenBiosystems provides) with XbaI and EcoRI (NEB), the plasmid that contains Insert Fragment utilizes Fugene6 (Roche) transfection reagent transient transfection to 293T cell (ATCC), cells and supernatant is according to (Yan, Lin et al.2003) the monoclonal antibody AA98 antigen purification method purifying of describing obtains the sCD146-AP fusion rotein.
Utilize ELISA to detect the combination of antibody and recombinant protein, simple method is as follows.
1) prepare Trx-His (its aminoacid sequence is shown in SEQ ID NO:7): according among the embodiment 2 about expressing the method for D3 and D5 albumen, be about to pET32a (Novagen) and transform B121 (DE3) competent cell, utilize the IPTG abduction delivering, through the Ni column purification.Spend the night with 4 ℃ of enzymolysis of Thrombin proteolytic enzyme (15U enzyme/mg albumen, Sigma provides), use afterwards the Ni-NTA purifying with the Trx-His of 6xHis label again, purifying D3 and D5 protein are seemingly among method and the embodiment 2.
2) prepare MBP-His (its aminoacid sequence is shown in SEQ ID NO:8): according among the embodiment 2 about expressing the method for D3 and D5 albumen, be about to HMV6g carrier (the carrier collection of illustrative plates as shown in figure 11) and transform B121 (DE3) competent cell, utilize the IPTG abduction delivering, through the Ni column purification.Spend the night with 4 ℃ of enzymolysis of Tev proteolytic enzyme (1 μ g enzyme/mg albumen, Invitrogen provides), use afterwards the Ni-NTA purifying with the MBP-His of 6xHis label again, method and embodiment 2 purifying D3 and D5 protein are seemingly.
3) on different holes on the elisa plate, be coated with respectively 1 μ g/ml antigen (His-rhCD146, sCD146-AP, Trx-His and MBP-His), 50 μ l/ holes, 4 ℃ of coated spending the night; With 2%BSA/PBS (Ameresco) room temperature sealing 2 hours, use afterwards antibody (the ascites AA1-5 of preparation and AA7) the 50 μ l/ pore chamber temperature of dilution in 1: 2000 to hatch onboard 2 hours, then wash 5 times with PBST, PBS washes 1 time.HRP coupling sheep anti-mouse igg (Santa Cruz) incubated at room of two anti-usefulness dilution in 1: 5,000 1 hour, PBST washes 5 times, and PBS washes 1 time.With tmb substrate (200ng/ml TMB (Ameresco), 0.03%H 2O 2(Beijing chemical reagents corporation), pH4.5) 100 μ l/ holes colour developing, sulfuric acid stops.The result as shown in Figure 1, AA1-AA5 and AA7 can both be very strong in conjunction with recombinant expressed CD146 protein extracellular: His-rhCD146 and sCD146-AP, yet the but Trx (Trx-His) that merges of debond 6xHis-tag and the mannose-binding protein (MBP-His) of 6xHis-tag fusion.This has confirmed the equal specific recognition CD146 albumen of this six strain antibody rather than His-tag.Use therein AA6 is as the negative control of experiment.
Embodiment 2: the epitope of monoclonal antibody AA1-AA5 and AA7 is identified.
Use recombinant expressed respectively five domain proteins of people source CD146 extracellular region and the method for immunoblotting, identified the epitope of six strain antibodies described in the present invention.
As shown in Figure 2, structural domain 1-5 represents respectively CD146 five structural domains of V-V-C2-C2-C2 to the cross-film district outside born of the same parents.Overlapping sequence is designed to prevent that possible epi-position from losing.Structural domain 1 (sequence is the amino acid of the position 24-145 in the people CD146 sequence of SEQ ID NO:1, shown in SEQ ID NO:9) is cloned on the pET30a (Novagen), and what express is that His6-Stag merges His6-S-D1 albumen.(sequence is the amino acid of the position 128-248 in the people CD146 sequence of SEQ ID NO:1 to structural domain 2, shown in SEQ ID NO:10), (sequence is the amino acid of the position 233-335 in the people CD146 sequence of SEQ ID NO:1 to structural domain 3, shown in SEQ IDNO:11), (sequence is the amino acid of the position 313-442 in the people CD146 sequence of SEQ ID NO:1 to structural domain 4, shown in SEQ ID NO:12) and structural domain 5 (sequence is the amino acid of the position 400-560 in the people CD146 sequence of SEQ IDNO:1, shown in SEQ ID NO:13) be cloned in respectively on the pET32a (Novagen), that express is respectively the Trx-His6-S-D2 that Trx-His6-S tag merges, Trx-His6-S-D3, Trx-His6-S-D4 and Trx-His6-S-D5.
Clone's process is summarized as follows: with corresponding pair of primers D1S and D1A (being used for structural domain 1), D2S and D2A (being used for structural domain 2), D3S and D3A (being used for structural domain 3), D4S and D4A (being used for structural domain 4), D5S and D5A (being used for structural domain 5), and template pcDNA3.1 (-) b-CD146 is (by deriving from Johnson with sequence shown in SEQ ID NO:2, the CD146 cDNA in J.P laboratory utilizes restriction enzyme EcoR I and BamH I (NEB) to be cloned into pcDNA3.1 (-) b carrier (Invitrogen) and obtains) do the PCR reaction, condition be 95 ℃ 5 minutes; 95 ℃ 45 seconds, 56 ℃ 40 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 5 minutes.Amplify respectively the nucleotide sequence of coding structure territory 1-5.
Primer is listed as follows, and the positive-sense strand primer is with Nco I restriction enzyme site (shown in underscore), and the antisense strand primer is with Hind III restriction enzyme site (shown in underscore):
The primer title Primer sequence 5 ' to 3 '
D1S 5’catg CCATGGTGCCCGGAGAGGCTGAG 3’(SEQ ID
NO：14)
D1A 5’AAGCTTttaGGGGTTGACCTGGATGTTTGGC 3’(SEQ ID NO：15)
D2S 5’CCATGGgtATCCAGCTCCGCGTCTACAAAG 3’(SEQ ID NO：16)
D2A 5’AAGCTTttaCGGGTAGAAAACAGGGACGGTG 3’(SEQ ID NO：17)
D3S 5’CCATGGgtAACCACATGAAGGAGTCCAGGGAAG 3’ (SEQ ID NO：18)
D3A 5’AAGCTTttaTGGTTCACTCAGCAGCGATATCATG 3 (SEQ ID NO：19)
D4S 5’CCATGGaaCACAGTGGGCGCTATGAATG 3’(SEQ ID NO：20)
D4A 5’AAGCTTttaCACCCACACCTTCCTCTCCTTG 3’(SEQ ID NO：21)
D5S 5’CCATGGagGCAGGAGGCGGCTATCG3’(SEQ ID NO：22)
D5A 5’AAGCTTttaGACCACGCCCCGGCTCTC 3’(SEQ ID NO：23)
The PCR product separates with 2% sepharose (Biowest Agarose), the sepharose DNA that provides with Tiangen company reclaims test kit (production code member: DP209-03) reclaim the PCR product, then the pMD18 T-simple kit that provides with Tara company is connected to fragment on the T carrier (carrier and enzyme are provided in the test kit), and transforms Top10 competent cell (Tiangen provides).Be added with the mono-clonal D1-T (having inserted the nucleotide sequence of structural domain 1) that grows on the LB culture plate of penbritin, D2-T (having inserted the nucleotide sequence of structural domain 2), D3-T (having inserted the nucleotide sequence of structural domain 3), D4-T (having inserted the nucleotide sequence of structural domain 4) and D5-T (having inserted the nucleotide sequence of structural domain 5), use with 37 ℃ of the LB culture medium culturing of penbritin and spend the night, and the little extraction reagent kit of plasmid that provides with Tiangen (production code member: DP103-03) extracting plasmid, with Nco I and Hind III (NEB) double digestion pET30a, pET32a, the plasmid (namely having inserted respectively the T carrier of the nucleotide sequence of structural domain 1-5) that extracts.Use afterwards 2% sepharose (Biowest Agarose) to separate, the sepharose DNA that provides with Tiangen company reclaims test kit, and (production code member: the enzyme that DP209-03) reclaims the T carrier is cut the plasmid fragment of small segment (being the nucleotide sequence of structural domain 1-5) and pET30a and the incision of pET32a enzyme.The enzyme of D1-T is cut small segment and is connected with the plasmid fragment that the pET30a enzyme cuts, D2-T, D3-T, the enzyme of D4-T and D5-T cut small segment and are connected with the plasmid fragment that the pET32a enzyme cuts, the T4 dna ligase that provides with the NEB company reaction that connects, 16 ℃ of connections are spent the night.Transform Top10 competent cell (Tiangen), pET30a-D1 screens with the semi-solid LB culture plate that contains kantlex (50ng/ml), pET32a-D2, pET32a-D3, pET32a-D4, and pET32a-D5 screens with the semi-solid LB culture plate that contains ammonia benzyl mycin (100ng/ml).The mono-clonal that incubated overnight obtains is respectively with containing antibiotic LB culture medium culturing, and the extracting plasmid, and the plasmid of acquisition is used for conversion B121 (DE3) competent cell (Tiangen).
Recombinant protein His6-S-D1, Trx-His6-S-D2, Trx-His6-S-D3, the abduction delivering of Trx-His6-S-D4 and Trx-His6-S-D5: transformed pET30a-D1, pET32a-D2, pET32a-D3, pET32a-D4, with B121 (DE3) mono-clonal of pET32a-D5,37 ℃ of overnight incubation in containing corresponding antibiotic 5mlLB nutrient solution.The bacterium that spends the night is seeded to 37 ℃ of cultivations in the corresponding fresh substratum at 1: 100, treat bacterial growth to OD600 be 0.6 o'clock, added 37 ℃ of abduction deliverings of 1mM IPTG (Ameresco) 6 hours or 16 ℃ of 0.2mM IPTG induce and spend the night.
D1, D2 and D4 present the inclusion body state by after the IPTG abduction delivering, and be soluble.Inclusion body purification and washing methods are as follows: thalline is resuspended in 25mM Tris-HCl (pH7.4), ultrasonic 2 * 99 circulations of 300W, and each circulating ultrasonic 4 seconds suspends 8 seconds, totally 12 seconds.Centrifugal 30 minutes of 4 ℃ of 12000g.Inclusion body precipitation is resuspended in the solution 1 (2.5M NaCl), and 4 ℃ were stirred 30 minutes, centrifugal 30 minutes of 4 ℃ of 12000g.Inclusion body precipitation is resuspended in the solution 2 (0.5%Triton X-100,10mMEDTA, pH8.0), and 4 ℃ were stirred 30 minutes, centrifugal 30 minutes of 4 ℃ of 12000g.Inclusion body precipitation is resuspended in the solution 3 (2M Urea, 50mM Tris, 1mM EDTA, pH8.0), and 4 ℃ were stirred 30 minutes, centrifugal 30 minutes of 4 ℃ of 12000g.Subsequently, solubilization of inclusion bodies is in solution 4 (8M Urea, 25mMTris, 150mM NaCl, 25mM DTT pH8.0).
D3 and D5 present solvable state by after the IPTG abduction delivering, use the Ni column purification.Concrete grammar is as follows: thalline is resuspended in 25mM Tris-HCl (pH7.4), ultrasonic 2 * 99 circulations of 300W, and each circulating ultrasonic 4 seconds suspends 8 seconds, totally 12 seconds.Centrifugal 30 minutes of 4 ℃ of 12000g collect supernatant, cross the 0.45mm filter membrane and remove impurity.Ni-NTA sepharose 6 Fast Flow (GE HealthCare) fill out post, after the ultrapure water washing, in 50mM Tris-HCl (pH7.4), balance in the balanced solution of 150mM NaCl.The centrifugal solvable supernatant loading hanging column that then will contain the albumen of His6-tag is washed post with washing soln (50mM Tris-HCl, pH7.4,150mM NaCl, 50mM imidazoles).Albumen on last elute soln (50mM Tris-HCl, pH7.4,150mM NaCl, 300mM imidazoles) the wash-out post.
Utilize immunoblot experiment to identify the epitope that AA1-AA5 and AA7 identify.Simple procedure is as follows: separate the D1-D5 albumen of Prokaryotic expression, purification with 15%SDS-PAGE, then use the half-dried transfer printing instrument of Bio-rad with the protein delivery on the glue to nitrocellulose filter.Sealed 2 hours with the 5% skimmed milk room temperature that is dissolved among the PBST, then use 4 ℃ of overnight incubation of primary antibodie solution (AA1-AA5 and AA7) of above-mentioned 5% skimmed milk/PBST dilution (1: 2000).Then PBST washes 5 times, resists with two of above-mentioned 5% skimmed milk/PBST dilution (1: 2000) that (sheep anti-mouse igg antibody of horseradish peroxidase, Pierce) the solution incubated at room is 1 hour.PBST washes after 5 times, with the chromogenic substrate (production code member: 34076,400 μ l 3%H of Pierce company 2O 2+ 400 μ l Luminol solution/miniblot) colour developing.In the experiment, utilize respectively AA1, AA2, AA3, AA4, AA5 and AA7 are as primary antibodie, the sheep anti-mouse igg of horseradish peroxidase (Pierce) is anti-as two, detect different antibodies and recombinant expressed five structural domain D1-5 of CD146 extracellular region in conjunction with activity.As shown in Figure 3, AA1 and AA2 be only in conjunction with D1, and the D2 that debond is adjacent has confirmed that its epitope is between the position 24-128 in the people CD146 sequence that sequence is SEQ ID NO:1, and concrete sequence is shown in SEQ ID NO:24.AA3-5 and AA7 be only in conjunction with D4, and the D3 that debond is adjacent and D5 have confirmed that its epitope is between the position 335-400 in the people CD146 sequence that sequence is SEQ ID NO:1, and concrete sequence is shown in SEQ ID NO:25.
Based on different epitopes this six strain antibody is divided into two classes, V1 class and C2-2 class.Because the epitope of AA1 and AA2 is positioned at first IgV spline structure territory, be classified as the V1 class, and AA3, AA4, the epitope of AA5 and AA7 are positioned at second IgC2 spline structure territory, so be classified as the C2-2 class.
Embodiment 3: utilize monoclonal antibody AA1-AA5 and AA7 to detect people CD146
Anti-human CD146 mouse monoclonal antibody of the present invention can and be organized level detection people CD146 albumen at molecule, cell.
In the whole-cell protein immunoblot experiment, AA1-AA5 and AA7 all can identify the CD146 albumen of reduction and non-reduced two states.Concrete experimental technique is as follows: the people source melanoma cell A375 (ATCC) that collects high expression level CD146, with twice of the PBS washed cell of precooling, centrifugal 5 minutes of 4 ℃ of 800rpm, cell precipitation lysate (Tris-HCl 50mM pH8.0, NaCl150mM, EDTA 1mM, NP-40 1%, Glycerol 10%, PMSF 100 μ g/ml) lysing cell, centrifugal 15 minutes of 4 ℃ of 12000g collect supernatant, add respectively sample-loading buffer (the 5x sample-loading buffer: 0.313MTris-HCl that contains DTT (final concentration 100mM) (dithiothreitol (DTT)) and do not contain DTT, pH6.8,10%SDS, 0.05% tetrabromophenol sulfonphthalein, 50% glycerine), 100 ℃ are boiled sample.10%SDS-PAGE separates whole-cell protein, and half-dried electrotransfer is to nitrocellulose filter afterwards.After the sealing of 5% skimmed milk, using respectively AA1-AA5 and AA7 to hatch film as 4 ℃ of primary antibodies (usefulness confining liquid 5% skimmed milk/TBST dilution in 1: 10000) spends the night, then the sheep anti-mouse igg two with horseradish peroxidase resists (Pierce, confining liquid 5% skimmed milk/TBST dilution in 1: 2000) incubated at room is 1 hour, chromogenic substrate (the production code member: 34076,400 μ l3%H that provides with Pierce company 2O 2+ 400 μ l Luminol solution/miniblot) detect.As shown in Figure 4, AA1-AA5 and AA7 can be at the CD146 protein molecular of molecular level in conjunction with the A375 expression.
Use the Flow cytometry means, confirmation V1 antibody-like can be identified the CD146 albumen of surface of cell membrane in the viable cell level, and the C2-2 antibody-like can not be identified.Concrete experimental technique is as follows: collect the people source melanoma cell A375 (the same) of high expression level CD146, and with the PBS washed cell twice that contains 0.3%BSA of precooling, every all over washing centrifugal 5 minutes of 4 ℃ of 800rpm afterwards.With sheep blood serum diluent (usefulness PBS diluted in 1: 20) closing cell, add afterwards AA1-AA5 and AA7, and positive control AA98 was hatched on ice 40 minutes as primary antibodie (PBS dilution in 1: 2000).With twice of the PBS washed cell that contains 0.3%BSA of precooling, add the sheep anti-mouse igg of FITC coupling as two anti-(Sigma, PBS dilution in 1: 300), incubated at room 20 minutes detects (BD Bioscience) with BD FACSCalibur flow cytometry system.The result as shown in Figure 5, AA1 and AA2 are the CD146 molecule that the V1 antibody-like can be expressed in conjunction with A375, with negative control mIgG (use as primary antibodie with mouse IgG, Sigma provides) obvious difference be arranged.And AA3-5 and AA7 are the C2-2 antibody-like, can not be in conjunction with the CD146 molecule of the native state on the viable cell A375 film, and possible reason is the epitope of C2-2 class identification is not exposed to the CD146 protein molecular under native state surface.
Equally, utilize immunofluorescence technique experiment, also only have the V1 antibody-like can identify the CD146 molecule of cell levels, and the C2-2 antibody-like can not, AA98 is as the positive control of experiment, mIgG is as negative control, as shown in Figure 6.Experiment has confirmed that also the CD146 molecule mainly is distributed in surface of cell membrane.Experimentation is summarized as follows: fixedly be longer than the A375 cell 1 minute of 24 orifice plates with 1: 1 acetone/methanol of precooling, afterwards the PBS washed twice.In 37 ℃ of closing cells 30 minutes, PBS washed twice with 2% sheep blood serum (China fir Golden Bridge in Beijing).Then add the primary antibodie (AA1-AA5 and AA7, AA98 and mIgG) of 100 μ l PBS dilution in 1: 2000, hatched 2 hours for 37 ℃.After the PBS washing, anti-ly hatched 1 hour with 37 ℃ in cell as two with the sheep anti-mouse igg (Sigma) of the FITC coupling of PBS dilution in 1: 300.PBS washes, microscopy.
The V1 antibody-like can be used for the natural CD146 molecule of enrichment solution.Utilize the technology of immunoprecipitation, the V1 antibody-like can in conjunction with and the enrichment of cell lysate in CD146 albumen.Concrete method is as follows: 1 * 10 7The cracking 30 minutes in the above-mentioned lysate of 0.6mL ice precooling of A375 (the same) cell, centrifugal 15 minutes of 4 ℃ of 12000g collect supernatant.Add first the proteinA-Agarose (Santa Cruz) of 20 μ l 50%slurry (50% mix with PBS or TE), carry out preclearing.Then add respectively 2 μ gAA1-AA5 and AA7,4 ℃ of overnight incubation.The protein A-Agarose capture antigen-antibody complex that adds afterwards 20 μ l 50%slurry with PBS washing precipitation three times, adds 40 μ l 1x sample-loading buffers (with 5 times of above-mentioned 5x sample-loading buffer dilutions), and 100 ℃ of heating were boiled sample 10 minutes.Albumen precipitation detects (experimental technique is same as above) with the method for immunoblotting, and AA98 is as detecting primary antibodie.As shown in Figure 7, AA1 and AA2 can precipitate the CD146 albumen in the enrichment A375 lysate, and the C2-2 antibody-like can not.
Utilize immunohistochemistry technology, the V1 antibody-like can detect the CD146 protein molecular of organizing level.Specific practice is as follows: human umbilical tissue carries out frozen section with OCT (frozen tissue embedding liquid, Sakura provides) embedding.Section is fixed 5 minutes in the acetone of precooling, then containing 0.3%H 2O 2Methyl alcohol in the room temperature black out hatch the interference of removing endogenous peroxydase in 30 minutes.After the PBS washing three times, with 5% horse serum (China fir Golden Bridge in Beijing, PBS dilutes horse serum) the sealing section, then containing AA1-AA5 respectively, AA7,4 ℃ of overnight incubation in the primary antibodie diluent of AA98 and anti-CD31 antibody (Santa Cruz) (confining liquid dilution in 1: 2000).Afterwards with the biotin labeled two anti-(sheep anti-mouse iggs of vitamin H coupling, Vector company, the dilution of 1: 1000 confining liquid) and the streptavidin of horseradish peroxidase resist (Vector as three, PBS dilution in 1: 1000), hatch tissue slice for 37 ℃, use at last DAB colouring reagents box (China fir Golden Bridge in Beijing, the production code member: ZLI-9033) colour developing of fresh configuration.Develop the color and redye nucleus with Hematorylin after complete.As shown in Figure 8, anti-CD31 antibody labeling vascular endothelial cell (as shown by arrows), AA1 and AA2 can organize level to identify the CD146 molecule (as shown by arrows) of umbilical vein endotheliocyte and vascular smooth muscle cell distribution, and the C2-2 antibody-like can not.AA98 is as the positive control (as shown by arrows) of experiment.
Embodiment 4: highly sensitive double antibodies sandwich ELISA detects human soluble CD14 6 methods
Utilize AA98 described in anti-human CD146 antibody A A1 described in the present invention and the patent of invention ZL 991075862, the present invention has developed a kind of highly sensitive double antibodies sandwich ELISA method, detects the sMe1-CAM in the serum.Main method is that (experiment about AA2 is identical with AA1, therefore omit) as capture antigen, the AA98 of vitamin H (Biotin) mark is as detectable antigens, the sCD146-AP of eukaryotic expression detects the sMe1-CAM albumen in normal people and the Serum of Patients With Autoimmune Diseases serum as standard substance.Experiment confirm, the sandwich ELISA of the present invention's exploitation has higher sensitivity and wider linearity range.Utilize this method also to confirm, the CD146 protein content among autoimmune disease such as the systemic vasculitis patients serum has remarkable rising than the healthy volunteer.
Concrete experimental technique is as follows:
1. sample source and clinical data: healthy volunteer's blood plasma is from BJ Red Cross Blood Center, and vasculitis patient's blood plasma is from the loyal hospital of peace.
2. concrete steps:
1) elisa plate coated antibody: antibody A A1 is diluted to 1 μ g/ml with 0.02M PB (pH 7.25), 4 ℃ of coated the spending the night in 50 μ l/ holes.
2) sealing non-specific binding site: 200 μ l/ hole 2%BSA/PBS, incubated at room 2 hours.
3) sample is hatched: the sCD146-AP gradient of concentration known is used for the drawing standard curve with confining liquid (2%BSA/PBS) dilution, and the concentration after the dilution is respectively 5,10,20,40,80,160,320ng/ml.20 times of test serum dilutions, applied sample amount 50 μ l, confining liquid replace sample as negative control.Each sample is established twice repetition.Incubated at room 2 hours.
4) PBST is 5 times, 1 flush away non-specific binding of PBS.
5) detect antibody incubation: detect antibody A A98-vitamin H (vitamin H that is used for mark AA98 is provided by Pierce company) and be diluted to 0.5 μ g/ml with 2%BSA/PB, 50 μ l/ pore chamber temperature were hatched 2 hours.
6) PBST is 5 times, 1 flush away non-specific binding of PBS.
7) ELIAS secondary antibody amplifying signal: add the streptavidin-horseradish peroxidase (Vector, 1: 3000) of suitable concn, 50 μ l/ holes, incubated at room 1 hour.
8) PBST is 5 times, 1 flush away non-specific binding of PBS.
9) color reaction: add substrate (200ng/ml TMB (Ameresco), 0.03%H 2O 2(Beijing chemical reagents corporation), pH4.5) colour developing, 100 μ l/ holes, 37 ℃ 15 minutes, add 50 μ l/ hole 2M H 2SO 4Termination reaction, microplate reader 450nm reading.
10) software statistics result: the software analysis light absorption value, according to standard sCD146-AP concentration drawing standard curve.
11) calculate the concentration of testing sample according to typical curve.
3. interpretation of result:
1) utilizes the concentration production standard curve of known sCD146-AP.The concentration of sCD146-AP is 5～320ng/ml after the dilution, and drawing standard curve such as Fig. 9 show.Draw as calculated the method sensing range and in 5～80ng/ml, meet linearity.Get sCD146-AP concentration 5～80ng/ml resulting typical curve (R that maps 2=0.9981), as shown in figure 10.
2) analyze sCD146 content in healthy volunteer and the systemic vasculitis patient blood plasma.SCD146 content is 154.7ng/ml in healthy volunteer's blood plasma, and vasculitis patient blood plasma sCD146 content reaches 383.9ng/ml, and the two obvious difference is as shown in table 2.
Table 2: utilize sMe1-CAM content among healthy volunteer that sandwich ELISA detects and the systemic vasculitis patients serum.
Test sample OD450 Diluent sCD146 concentration (ng/ml) Stoste sCD146 concentration (ng/ml)
The healthy volunteer 0.133 7.73191 154.7
The systemic vasculitis patient 0.3175 19.1925 383.9
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The IB080807-sequence table
SEQUENCE LISTING
＜110〉Institute of Biophysics, Academia Sinica
＜120〉monoclonal antibody of anti-human CD146 comprises its composition, detects the method for sMe1-CAM
<130>IB080807
<160>42
<170>PatentIn version 3.1
<210>1
<211>645
<212>PRT
＜213〉people
<400>1
Met Gly Leu Pro Arg Leu Val Cys Ala Phe Leu Leu Ala Ala Cys Cys
1 5 10 15
Cys Cys Pro Arg Val Ala Gly Val Pro Gly Glu Ala Glu Gln Pro Ala
20 25 30
Pro Glu Leu Val Glu Val Glu Val Gly Ser Thr Ala Leu Leu Lys Cys
35 40 45
Gly Leu Ser Gln Ser Gln Gly Asn Leu Ser His Val Asp Trp Phe Ser
50 55 60
Val His Lys Glu Lys Arg Thr Leu Ile Phe Arg Val Arg Gln Gly Gln
65 70 75 80
Gly Gln Ser Glu Pro Gly Glu Tyr Glu Gln Arg Leu Ser Leu Gln Asp
85 90 95
Arg Gly Ala Thr Leu Ala Leu Thr Gln Val Thr Pro Gln Asp Glu Arg
100 105 110
Ile Phe Leu Cys Gln Gly Lys Arg Pro Arg Ser Gln Glu Tyr Arg Ile
115 120 125
Gln Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro Asn Ile Gln Val Asn
130 135 140
Pro Leu Gly Ile Pro Val Asn Ser Lys Glu Pro Glu Glu Val Ala Thr
145 150 155 160
Cys Val Gly Arg Asn Gly Tyr Pro Ile Pro Gln Val Ile Trp Tyr Lys
165 170 175
Asn Gly Arg Pro Leu Lys Glu Glu Lys Asn Arg Val His Ile Gln Ser
180 185 190
Ser Gln Thr Val Glu Ser Ser Gly Leu Tyr Thr Leu Gln Ser Ile Leu
195 200 205
Lys Ala Gln Leu Val Lys Glu Asp Lys Asp Ala Gln Phe Tyr Cys Glu
210 215 220
Leu Asn Tyr Arg Leu Pro Ser Gly Asn His Met Lys Glu Ser Arg Glu
225 230 235 240
Val Thr Val Pro Val Phe Tyr Pro Thr Glu Lys Val Trp Leu Glu Val
245 250 255
Glu Pro Val Gly Met Leu Lys Glu Gly Asp Arg Val Glu Ile Arg Cys
260 265 270
Leu Ala Asp Gly Asn Pro Pro Pro His Phe Ser Ile Ser Lys Gln Asn
275 280 285
Pro Ser Thr Arg Glu Ala Glu Glu Glu Thr Thr Asn Asp Asn Gly Val
290 295 300
Leu Val Leu Glu Pro Ala Arg Lys Glu His Ser Gly Arg Tyr Glu Cys
305 310 315 320
Gln Gly Leu Asp Leu Asp Thr Met Ile Ser Leu Leu Ser Glu Pro Gln
325 330 335
Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro Ala Ala
340 345 350
Pro Glu Arg Gln Glu Gly Ser Ser Leu Thr Leu Thr Cys Glu Ala Glu
355 360 365
Ser Ser Gln Asp Leu Glu Phe Gln Trp Leu Arg Glu Glu Thr Gly Gln
370 375 380
Val Leu Glu Arg Gly Pro Val Leu Gln Leu His Asp Leu Lys Arg Glu
385 390 395 400
Ala Gly Gly Gly Tyr Arg Cys Val Ala Ser Val Pro Ser Ile Pro Gly
405 410 415
Leu Asn Arg Thr Gln Leu Val Asn Val Ala Ile Phe Gly Pro Pro Trp
420 425 430
Met Ala Phe Lys Glu Arg Lys Val Trp Val Lys Glu Asn Met Val Leu
435 440 445
Asn Leu Ser Cys Glu Ala Ser Gly His Pro Arg Pro Thr Ile Ser Trp
450 455 460
Asn Val Asn Gly Thr Ala Ser Glu Gln Asp Gln Asp Pro Gln Arg Val
465 470 475 480
Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu Thr Gly
485 490 495
Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn Ser Ile Leu Phe
500 505 510
Leu Glu Leu Val Asn Leu Thr Thr Leu Thr Pro Asp Ser Asn Thr Thr
515 520 525
Thr Gly Leu Ser Thr Ser Thr Ala Ser Pro His Thr Arg Ala Asn Ser
530 535 540
Thr Ser Thr Glu Arg Lys Leu Pro Glu Pro Glu Ser Arg Gly Val Val
545 550 555 560
Ile Val Ala Val Ile Val Cys Ile Leu Val Leu Ala Val Leu Gly Ala
565 570 575
Val Leu Tyr Phe Leu Tyr Lys Lys Gly Lys Leu Pro Cys Arg Arg Ser
580 585 590
Gly Lys Gln Glu Ile Thr Leu Pro Pro Ser Arg Lys Ser Glu Leu Val
595 600 605
Val Glu Val Lys Ser Asp Lys Leu Pro Glu Glu Met Gly Leu Leu Gln
610 615 620
Gly Ser Ser Gly Asp Lys Arg Ala Pro Gly Asp Gln Gly Glu Lys Tys
625 630 635 640
Ile Asp Leu Arg His
645
<210>2
<211>1941
<212>DNA
＜213〉people
<400>2
atggggcttc ccaggctggt ctgcgccttc ttgctcgccg cctgctgctg ctgtcctcgc 60
gtcgcgggtg tgcccggaga ggctgagcag cctgcgcctg agctggtgga ggtggaagtg 120
ggcagcacag cccttctgaa gtgcggcctc tcccagtccc aaggcaacct cagccatgtc 180
gactggtttt ctgtccacaa ggagaagcgg acgctcatct tccgtgtgcg ccagggccag 240
ggccagagcg aacctgggga gtacgagcag cggctcagcc tccaggacag aggggctact 300
ctggccctga ctcaagtcac cccccaagac gagcgcatct tcttgtgcca gggcaagcgc 360
cctcggtccc aggagtaccg catccagctc cgcgtctaca aagctccgga ggagccaaac 420
atccaggtca accccctggg catccctgtg aacagtaagg agcctgagga ggtcgctacc 480
tgtgtaggga ggaacgggta ccccattcct caagtcatct ggtacaagaa tggccggcct 540
ctgaaggagg agaagaaccg ggtccacatt cagtcgtccc agactgtgga gtcgagtggt 600
ttgtacacct tgcagagtat tctgaaggca cagctggtta aagaagacaa agatgcccag 660
ttttactgtg agctcaacta ccggctgccc agtgggaacc acatgaagga gtccagggaa 720
gtcaccgtcc ctgttttcta cccgacagaa aaagtgtggc tggaagtgga gcccgtggga 780
atgctgaagg aaggggaccg cgtggaaatc aggtgtttgg ctgatggcaa ccctccacca 840
cacttcagca tcagcaagca gaaccccagc accagggagg cagaggaaga gacaaccaac 900
gacaacgggg tcctggtgct ggagcctgcc cggaaggaac acagtgggcg ctatgaatgt 960
cagggcctgg acttggacac catgatatcg ctgctgagtg aaccacagga actactggtg 1020
aactatgtgt ctgacgtccg agtgagtccc gcagcccctg agagacagga aggcagcagc 1080
ctcaccctga cctgtgaggc agagagtagc caggacctcg agttccagtg gctgagagaa 1140
gagacaggcc aggtgctgga aagggggcct gtgcttcagt tgcatgacct gaaacgggag 1200
gcaggaggcg gctatcgctg cgtggcgtct gtgcccagca tacccggcct gaaccgcaca 1260
cagctggtca acgtggccat ttttggcccc ccttggatgg cattcaagga gaggaaggtg 1320
tgggtgaaag agaatatggt gttgaatctg tcttgtgaag cgtcagggca cccccggccc 1380
accatctcct ggaacgtcaa cggcacggca agtgaacaag accaagatcc acagcgagtc 1440
ctgagcaccc tgaatgtcct cgtgaccccg gagctgttgg agacaggtgt tgaatgcacg 1500
gcctccaacg acctgggcaa aaacaccagc atcctcttcc tggagctggt caatttaacc 1560
accctcacac cagactccaa cacaaccact ggcctcagca cttccactgc cagtcctcat 1620
accagagcca acagcacctc cacagagaga aagctgccgg agccggagag ccggggcgtg 1680
gtcatcgtgg ctgtgattgt gtgcatcctg gtcctggcgg tgctgggcgc tgtcctctat 1740
ttcctctata agaagggcaa gctgccgtgc aggcgctcag ggaagcagga gatcacgcta 1800
cccccgtctc gtaagagcga acttgtagtt gaagttaagt cagataagct cccagaagag 1860
atgggcctcc tgcagggcag cagcggtgac aagagggctc cgggagacca gggagagaaa 1920
tacatcgatc tgaggcatta g 1941
<210>3
<211>471
<212>PRT
＜213〉artificial sequence
<400>3
Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr
1 5 10 15
Pro Val Thr Lys Ala Arg Thr Pro Glu Met Pro Val Leu Glu Asn Arg
20 25 30
Ala Ala Gln Gly Asp Ile Thr Ala Pro Gly Gly Ala Arg Arg Leu Thr
35 40 45
Gly Asp Gln Thr Ala Ala Leu Arg Asp Ser Leu Ser Asp Lys Pro Ala
50 55 60
Lys Asn Ile Ile Leu Leu Ile Gly Asp Gly Met Gly Asp Ser Glu Ile
65 70 75 80
Thr Ala Ala Arg Asn Tyr Ala Glu Gly Ala Gly Gly Phe Phe Lys Gly
85 90 95
Ile Asp Ala Leu Pro Leu Thr Gly Gln Tyr Thr His Tyr Ala Leu Asn
100 105 110
Lys Lys Thr Gly Lys Pro Asp Tyr Val Thr Asp Ser Ala Ala Ser Ala
115 120 125
Thr Ala Trp Ser Thr Gly Val Lys Thr Tyr Asn Gly Ala Leu Gly Val
130 135 140
Asp Ile His Glu Lys Asp His Pro Thr Ile Leu Glu Met Ala Lys Ala
145 150 155 160
Ala Gly Leu Ala Thr Gly Asn Val Ser Thr Ala Glu Leu Gln Asp Ala
165 170 175
Thr Pro Ala Ala Leu Val Ala His Val Thr Ser Arg Lys Cys Tyr Gly
180 185 190
Pro Ser Ala Thr Ser Glu Lys Cys Pro Gly Asn Ala Leu Glu Lys Gly
195 200 205
Gly Lys Gly Ser Ile Thr Glu Gln Leu Leu Asn Ala Arg Ala Asp Val
210 215 220
Thr Leu Gly Gly Gly Ala Lys Thr Phe Ala Glu Thr Ala Thr Ala Gly
225 230 235 240
Glu Trp Gln Gly Lys Thr Leu Arg Glu Gln Ala Gln Ala Arg Gly Tyr
245 250 255
Gln Leu Val Ser Asp Ala Ala Ser Leu Asn Ser Val Thr Glu Ala Asn
260 265 270
Gln Gln Lys Pro Leu Leu Gly Leu Phe Ala Asp Gly Asn Met Pro Val
275 280 285
Arg Trp Leu Gly Pro Lys Ala Thr Tyr His Gly Asn Ile Asp Lys Pro
290 295 300
Ala Val Thr Cys Thr Pro Asn Pro Gln Arg Asn Asp Ser Val Pro Thr
305 310 315 320
Leu Ala Gln Met Thr Asp Lys Ala Ile Glu Leu Leu Ser Lys Asn Glu
325 330 335
Lys Gly Phe Phe Leu Gln Val Glu Gly Ala Ser Ile Asp Lys Gln Asp
340 345 350
His Ala Ala Asn Pro Cys Gly Gln Ile Gly Glu Thr Val Asp Leu Asp
355 360 365
Glu Ala Val Gln Arg Ala Leu Glu Phe Ala Lys Lys Glu Gly Asn Thr
370 375 380
Leu Val Ile Val Thr Ala Asp His Ala His Ala Ser Gln Ile Val Ala
385 390 395 400
Pro Asp Thr Lys Ala Pro Gly Leu Thr Gln Ala Leu Asn Thr Lys Asp
405 410 415
Gly Ala Val Met Val Met Ser Tyr Gly Asn Ser Glu Glu Asp Ser Gln
420 425 430
Glu His Thr Gly Ser Gln Leu Arg Ile Ala Ala Tyr Gly Pro His Ala
435 440 445
Ala Asn Val Val Gly Leu Thr Asp Gln Thr Asp Leu Phe Tyr Thr Met
450 455 460
Lys Ala Ala Leu Gly Leu Lys
465 470
<210>4
<211>29
<212>DNA
＜213〉artificial sequence
<400>4
aacatatggt gcccggagag gctgagcag 29
<210>5
<211>29
<212>DNA
＜213〉artificial sequence
<400>5
aagcggccgc cagctttctc tctgtggag 29
<210>6
<211>3129
<212>DNA
(213〉artificial sequence
<400>6
atggggcttc ccaggctggt ctgcgccttc ttgctcgccg cctgctgctg ctgtcctcgc 60
gtcgcgggtg tgcccggaga ggctgagcag cctgcgcctg agctggtgga ggtggaagtg 120
ggcagcacag cccttctgaa gtgcggcctc tcccagtccc aaggcaacct cagccatgtc 180
gactggtttt ctgtccacaa ggagaagcgg acgctcatct tccgtgtgcg ccagggccag 240
ggccagagcg aacctgggga gtacgagcag cggctcagcc tccaggacag aggggctact 300
ctggccctga ctcaagtcac cccccaagac gagcgcatct tcttgtgcca gggcaagcgc 360
cctcggtccc aggagtaccg catccagctc cgcgtctaca aagctccgga ggagccaaac 420
atccaggtca accccctggg catccctgtg aacagtaagg agcctgagga ggtcgctacc 480
tgtgtaggga ggaacgggta ccccattcct caagtcatct ggtacaagaa tggccggcct 540
ctgaaggagg agaagaaccg ggtccacatt cagtcgtccc agactgtgga gtcgagtggt 600
ttgtacacct tgcagagtat tctgaaggca cagctggtta aagaagacaa agatgcccag 660
ttttactgtg agctcaacta ccggctgccc agtgggaacc acatgaagga gtccagggaa 720
gtcaccgtcc ctgttttcta cccgacagaa aaagtgtggc tggaagtgga gcccgtggga 780
atgctgaagg aaggggaccg cgtggaaatc aggtgtttgg ctgatggcaa ccctccacca 840
cacttcagca tcagcaagca gaaccccagc accagggagg cagaggaaga gacaaccaac 900
gacaacgggg tcctggtgct ggagcctgcc cggaaggaac acagtgggcg ctatgaatgt 960
cagggcctgg acttggacac catgatatcg ctgctgagtg aaccacagga actactggtg 1020
aactatgtgt ctgacgtccg agtgagtccc gcagcccctg agagacagga aggcagcagc 1080
ctcaccctga cctgtgaggc agagagtagc caggacctcg agttccagtg gctgagagaa 1140
gagacaggcc aggtgctgga aagggggcct gtgcttcagt tgcatgacct gaaacgggag 1200
gcaggaggcg gctatcgctg cgtggcgtct gtgcccagca tacccggcct gaaccgcaca 1260
cagctggtca acgtggccat ttttggcccc ccttggatgg cattcaagga gaggaaggtg 1320
tgggtgaaag agaatatggt gttgaatctg tcttgtgaag cgtcagggca cccccggccc 1380
accatctcct ggaacgtcaa cggcacggca agtgaacaag accaagatcc acagcgagtc 1440
ctgagcaccc tgaatgtcct cgtgaccccg gagctgttgg agacaggtgt tgaatgcacg 1500
gcctccaacg acctgggcaa aaacaccagc atcctcttcc tggagctggt caatttaacc 1560
accctcacac cagactccaa cacaaccact ggcctcagca cttccactgc cagtcctcat 1620
accagagcca acagcacctc cacagagaga aagctgccgg agccggagag ccggggcggc 1680
ggcggcagcg gcggcggcag cggcggcggc agcgtgaaac aaagcactat tgcactggca 1740
ctcttaccgt tactgtttac ccctgtgaca aaagcccgga caccagaaat gcctgttctg 1800
gaaaaccggg ctgctcaggg cgatattact gcacccggcg gtgctcgccg tttaacgggt 1860
gatcagactg ccgctctgcg tgattctctt agcgataaac ctgcaaaaaa tattattttg 1920
ctgattggcg atgggatggg ggactcggaa attactgccg cacgtaatta tgccgaaggt 1980
gcgggcggct tttttaaagg tatagatgcc ttaccgctta ccgggcaata cactcactat 2040
gcgctgaata aaaaaaccgg caaaccggac tacgtcaccg actcggctgc atcagcaacc 2100
gcctggtcaa ccggtgtcaa aacctataac ggcgcgctgg gcgtcgatat tcacgaaaaa 2160
gatcacccaa cgattctgga aatggcaaaa gccgcaggtc tggcgaccgg taacgtttct 2220
accgcagagt tgcaggatgc cacgcccgct gcgctggtgg cacatgtgac ctcgcgcaaa 2280
tgctacggtc cgagcgcgac cagtgaaaaa tgtccgggta acgctctgga aaaaggcgga 2340
aaaggatcga ttaccgaaca gctgcttaac gctcgtgccg acgttacgct tggcggcggc 2400
gcaaaaacct ttgctgaaac ggcaaccgct ggtgaatggc agggaaaaac gctgcgtgaa 2460
caggcacagg cgcgtggtta tcagttggtg agcgatgctg cctcactgaa ttcggtgacg 2520
gaagcgaatc agcaaaaacc cctgcttggc ctgtttgctg acggcaatat gccagtgcgc 2580
tggctaggac cgaaagcaac gtaccatggc aatatcgata agcccgcagt cacctgtacg 2640
ccaaatccgc aacgtaatga cagtgtacca accctggcgc agatgaccga caaagccatt 2700
gaattgttga gtaaaaatga gaaaggcttt ttcctgcaag ttgaaggtgc gtcaatcgat 2760
aaacaggatc atgctgcgaa tccttgtggg caaattggcg agacggtcga tctcgatgaa 2820
gccgtacaac gggcgctgga attcgctaaa aaggagggta acacgctggt catagtcacc 2880
gctgatcacg cccacgccag ccagattgtt gcgccggata ccaaagctcc gggcctcacc 2940
caggcgctaa ataccaaaga tggcgcagtg atggtgatga gttacgggaa ctccgaagag 3000
gattcacaag aacataccgg cagtcagttg cgtattgcgg cgtatggccc gcatgccgcc 3060
aatgttgttg gactgaccga ccagaccgat ctcttctaca ccatgaaagc cgctctgggg 3120
ctgaaataa 3129
<210>7
<211>129
<212>PRT
＜213〉artificial sequence
<400>7
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ale Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
115 120 125
Arg
<210>8
<211>386
<212>PRT
＜213〉artificial sequence
<400>8
Met Gly Ser Ser His His His His His His Gly Thr Lys Thr Glu Glu
1 5 10 15
Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu
20 25 30
Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
35 40 45
Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala
50 55 60
Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
65 70 75 80
Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala
85 90 95
Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn
100 105 110
Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
115 120 125
Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile
130 135 140
Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met
145 150 155 160
Glu Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
165 170 175
Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp
180 185 190
Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
195 200 205
Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile
210 215 220
Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly
225 230 235 240
Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val
245 250 255
Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
260 265 270
Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala
275 280 285
Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
290 295 300
Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu
305 310 315 320
Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala
325 330 335
Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp
340 345 350
Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr
355 360 365
Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Gly Thr Asp Tyr Asp Ile
370 375 380
Pro Thr
385
<210>9
<211>122
<212>PRT
＜213〉people
<400>9
Val Pro Gly Glu Ala Glu Gln Pro Ala Pro Glu Leu Val Glu Val Glu
1 5 10 15
Val Gly Ser Thr Ala Leu Leu Lys Cys Gly Leu Ser Gln Ser Gln Gly
20 25 30
Asn Leu Ser His Val Asp Trp Phe Ser Val His Lys Glu Lys Arg Thr
35 40 45
Leu Ile Phe Arg Val Arg Gln Gly Gln Gly Gln Ser Glu Pro Gly Glu
50 55 60
Tyr Glu Gln Arg Leu Ser Leu Gln Asp Arg Gly Ala Thr Leu Ala Leu
65 70 75 80
Thr Gln Val Thr Pro Gln Asp Glu Arg Ile Phe Leu Cys Gln Gly Lys
85 90 95
Arg Pro Arg Ser Gln Glu Tyr Arg Ile Gln Leu Arg Val Tyr Lys Ala
100 105 110
Pro Glu Glu Pro Asn Ile Gln Val Asn Pro
115 120
<210>10
<211>121
<212>PRT
＜213〉people
<400>10
Ile Gln Leu Arg Val Tyr Lys Ala Pro Glu Glu Pro Asn Ile Gln Val
1 5 10 15
Asn Pro Leu Gly Ile Pro Val Asn Ser Lys Glu Pro Glu Glu Val Ala
20 25 30
Thr Cys Val Gly Arg Asn Gly Tyr Pro Ile Pro Gln Val Ile Trp Tyr
35 40 45
Lys Asn Gly Arg Pro Leu Lys Glu Glu Lys Asn Arg Val His Ile Gln
50 55 60
Ser Ser Gln Thr Val Glu Ser Ser Gly Leu Tyr Thr Leu Gln Ser Ile
65 70 75 80
Leu Lys Ala Gln Leu Val Lys Glu Asp Lys Asp Ala Gln Phe Tyr Cys
85 90 95
Glu Leu Asn Tyr Arg Leu Pro Ser Gly Asn His Met Lys Glu Ser Arg
100 105 110
Glu Val Thr Val Pro Val Phe Tyr Pro
115 120
<210>11
<211>103
<212>PRT
＜213〉people
<400>11
Asn His Met Lys Glu Ser Arg Glu Val Thr Val Pro Val Phe Tyr Pro
1 5 10 15
Thr Glu Lys Val Trp Leu Glu Val Glu Pro Val Gly Met Leu Lys Glu
20 25 30
Gly Asp Arg Val Glu Ile Arg Cys Leu Ala Asp Gly Asn Pro Pro Pro
35 40 45
His Phe Ser Ile Ser Lys Gln Asn Pro Ser Thr Arg Glu Ala Glu Glu
50 55 60
Glu Thr Thr Asn Asp Asn Gly Val Leu Val Leu Glu Pro Ala Arg Lys
65 70 75 80
Glu His Ser Gly Arg Tyr Glu Cys Gln Gly Leu Asp Leu Asp Thr Met
85 90 95
Ile Ser Leu Leu Ser Glu Pro
100
<210>12
<211>130
<212>PRT
＜213〉people
<400>12
Glu His Ser Gly Arg Tyr Glu Cys Gly Gly Leu Asp Leu Asp Thr Met
1 5 10 15
Ile Ser Leu Leu Ser Glu Pro Glu Gln Leu Leu Val Asn Tyr Val Ser
20 25 30
Asp Val Arg Val Ser Pro Ala Ala Pro Glu Arg Gln Glu Gly Ser Ser
35 40 45
Leu Thr Leu Thr Cys Glu Ala Glu Ser Ser Gln Asp Leu Glu Phe Gln
50 55 60
Trp Leu Arg Glu Glu Thr Gly Gln Val Leu Glu Arg Gly Pro Val Leu
65 70 75 80
Gln Leu His Asp Leu Lys Arg Glu Ala Gly Gly Gly Tyr Arg Cys Val
85 90 95
Ala Ser Val Pro Ser Ile Pro Gly Leu Asn Arg Thr Gln Leu Val Asn
100 105 100
Val Ala Ile Phe Gly Pro Pro Trp Met Ala Phe Lys Glu Arg Lys Val
115 120 125
Trp Val
130
<210>13
<211>161
<212>PRT
＜213〉people
<400>13
Glu Ala Gly Gly Gly Tyr Arg Cys Val Ala Ser Val Pro Ser Ile Pro
1 5 10 15
Gly Leu Asn Arg Thr Gln Leu Val Asn Val Ala Ile Phe Gly Pro Pro
20 25 30
Trp Met Ala Phe Lys Glu Arg Lys Val Trp Val Lys Glu Asn Met Val
35 40 45
Leu Asn Leu Ser Cys Glu Ala Ser Gly His Pro Arg Pro Thr Ile Ser
50 55 60
Trp Asn Val Asn Gly Thr Ala Ser Glu Gln Asp Gln Asp Pro Gln Arg
65 70 75 80
Val Leu Ser Thr Leu Asn Val Leu Val Thr Pro Glu Leu Leu Glu Thr
85 90 95
Gly Val Glu Cys Thr Ala Ser Asn Asp Leu Gly Lys Asn Ser Ile Leu
100 105 110
Phe Leu Glu Leu Val Asn Leu Thr Thr Leu Thr Pro Asp Ser Asn Thr
115 120 125
Thr Thr Gly Leu Ser Thr Ser Thr Ala Ser Pro His Thr Arg Ala Asn
130 135 140
Ser Thr Ser Thr Glu Arg Lys Leu Pro Glu Pro Glu Ser Arg Gly Val
145 150 155 160
Val
<210>14
<211>27
<212>DNA
＜213〉artificial sequence
<400>14
catgccatgg tgcccggaga ggctgag 27
<210>15
<211>31
<212>DNA
＜213〉artificial sequence
<400>15
aagcttttag gggttgacct ggatgtttgg c 31
<210>16
<211>30
<212>DNA
＜213〉artificial sequence
<400>16
ccatgggtat ccagctccgc gtctacaaag 30
<210>17
<211>31
<212>DNA
＜213〉artificial sequence
<400>17
aagcttttac gggtagaaaa cagggacggt g 31
<210>18
<211>33
<212>DNA
＜213〉artificial sequence
<400>18
ccatgggtaa ccacatgaag gagtccaggg aag 33
<210>19
<211>34
<212>DNA
＜213〉artificial sequence
<400>19
aagcttttat ggttcactca gcagcgatat catg 34
<210>20
<211>28
<212>DNA
＜213〉artificial sequence
<400>20
ccatggaaca cagtgggcgc tatgaatg 28
<210>21
<211>31
<212>DNA
＜213〉artificial sequence
<400>21
aagcttttac acccacacct tcctctcctt g 31
<210>22
<211>25
<212>DNA
＜213〉artificial sequence
<400>22
ccatggaggc aggaggcggc tatcg 25
<210>23
<211>27
<212>DNA
＜213〉artificial sequence
<400>23
aagcttttag accacgcccc ggctctc 27
<210>24
<211>105
<212>PRT
＜213〉people
<400>24
Val Pro Gly Glu Ala Glu Gln Pro Ala Pro Leu Leu Val Glu Val Glu
1 5 10 15
Val Gly Ser Thr Ala Leu Leu Lys Cys Gly Leu Ser Gln Ser Gln Gly
20 25 30
Asn Leu Ser His Val Asp Trp Phe Ser Val His Lys Glu Lys Arg Thr
35 40 45
Leu Ile Phe Arg Val Arg Gln Gly Gln Gly Gln Ser Glu Pro Gly Glu
50 55 60
Tyr Glu Gln Arg Leu Ser Leu Gln Asp Arg Gly Ala Thr Leu Ala Leu
65 70 75 80
Thr Gln Val Thr Pro Gln Asp Glu Arg Ile Phe Leu Cys Gln Gly Lys
85 90 95
Arg Pro Arg Ser Gln Glu Tyr Arg Ile
100 105
<210>25
<211>66
<212>PRT
＜213〉people
<400>25
Pro Gln Glu Leu Leu Val Asn Tyr Val Ser Asp Val Arg Val Ser Pro
1 5 10 15
Ala Ala Pro Glu Arg Gln Glu Gly Ser Ser Leu Thr Leu Thr Cys Glu
20 25 30
Ala Glu Ser Ser Gln Asp Leu Glu Phe Gln Trp Leu Arg Glu Glu Thr
35 40 45
Gly Gln Val Leu Glu Arg Gly Pro Val Leu Gln Leu His Asp Leu Lys
50 55 60
Arg Glu
65
<210>26
<211>8
<212>PRT
＜213〉artificial sequence
<400>26
Gly Leu Thr Phe Thr Glu Tyr Thr
1 5
<210>27
<211>8
<212>PRT
＜213〉artificial sequence
<400>27
Ile Asn Pro Asn Asn Gly Gly Pro
1 5
<210>28
<211>9
<212>PRT
＜213〉artificial sequence
<400>28
Ala Arg Asn Gly Gly Asp Phe Ala Tyr
1 5
<210>29
<211>12
<212>PRT
＜213〉artificial sequence
<400>29
Gln Ser Leu Leu Tyr Ser Ser Asn Gln Glu Asn Tyr
1 5 10
<210>30
<211>3
<212>PRT
＜213〉artificial sequence
<400>30
Trp Ala Ser
1
<210>31
<211>9
<212>PRT
＜213〉artificial sequence
<400>31
Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr
1 5
<210>32
<211>116
<212>PRT
＜213〉artificial sequence
<400>32
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Leu Thr Phe Thr Glu Tyr
20 25 30
Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Thr Ile Asn Pro Asn Asn Gly Gly Pro Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Asn Gly Gly Asp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala
115
<210>33
<211>113
<212>PRT
＜213〉artificial sequence
<400>33
Thr Leu Val Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr
65 70 75 80
Ile Ser Thr Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Glu Leu Glu Leu
100 105 110
Lys
<210>34
<211>8
<212>PRT
＜213〉artificial sequence
<400>34
Gly Phe Thr Phe Ser Thr Tyr Ala
1 5
<210>35
<211>7
<212>PRT
＜213〉artificial sequence
<400>35
Ile Ser Ser Gly Ser Arg Thr
1 5
<210>36
<211>8
<212>PRT
＜213〉artificial sequence
<400>36
Val Arg Gly Pro Ala Phe Ala His
1 5
<210>37
<211>11
<212>PRT
＜213〉artificial sequence
<400>37
Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr
1 5 10
<210>38
<211>3
<212>PRT
＜213〉artificial sequence
<400>38
Leu Val Ser
1
<210>39
<211>8
<212>PRT
＜213〉artificial sequence
<400>39
Cys Gln Gly Thr His Phe Ser Thr
1 5
<210>40
<211>114
<212>PRT
＜213〉artificial sequence
<400>40
Glu Val Lys Leu Glu Glu Ser Gly Glu Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Ser Gly Ser Arg Thr Tyr Tyr Ser Asp Ser Val Lys
50 55 60
Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Arg Asn Asn Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val
85 90 95
Arg Gly Pro Ala Phe Ala His Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ala
<210>41
<211>111
<212>PRT
＜213〉artificial sequence
<400>41
Asp Ile Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Thr Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly
85 90 95
Thr His Phe Ser Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210>42
<211>12
<212>PRT
＜213〉artificial sequence
<400>42
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10

Claims (15)

1.SEQ the sequence shown in the amino-acid residue 24-145 of ID NO:1.
2. anti-human CD146 antibody is characterized in that the aminoacid sequence that it can specific recognition claim 1, and it is the hybridoma cell strain secretion of CGMCC NO.2310 by preserving number.
The antibody of the anti-human CD146 of claim 2 for the preparation of the treatment tumour pharmaceutical composition in application, wherein said tumour is melanoma, liver cancer, carcinoma of the pancreas.
4. the antibody of the anti-human CD146 of claim 2 is for the preparation of the application in the diagnostic reagent of the imaging level diagnosis of the human tumor of target CD146.
5. pharmaceutical composition that is used for the treatment of tumour, it comprises anti-human CD146 antibody and the pharmaceutical carrier of claim 2.
6. secrete the hybridoma cell strain of anti-human CD146 antibody, preserving number is CGMCC NO:2310.
7. the nucleic acid of the described anti-human CD146 antibody of the claim 2 of encoding.
8. one kind comprises the expression vector according to the nucleic acid of claim 7, and it can express described nucleic acid in protokaryon or eukaryotic host cell.
9. a protokaryon or eukaryotic host cell, it comprises the carrier according to claim 8.
10. method for preparing in conjunction with the antibody of people CD146 is characterized in that expressing in protokaryon or eukaryotic host cell according to the nucleic acid of the encoding antibody heavy chain of claim 7 and the nucleic acid of encoding antibody light chain, and reclaim described polypeptide from described cell.
11. the anti-human CD146 antibody of claim 2 is for the preparation of the application in the diagnostic reagent that detects people CD146, wherein said detection is undertaken by enzyme-linked immunosorbent assay.
12. the application of claim 11, wherein said enzyme-linked immunosorbent assay is sandwich enzyme-linked immunoabsorption, wherein with anti-human CD146 antibody claimed in claim 2 as capture antibody, with the mouse monoclonal antibody AA98 among the Chinese patent ZL991075862 as detecting antibody.
13. the application of claim 11 or 12, wherein said people CD146 is present in the human body fluid.
14. the application of claim 12, wherein said body fluid is tissue juice, serum, lymph liquid or cerebrospinal fluid.
15. the derivative of the antibody of the anti-human CD146 of claim 2, wherein said derivative are the product that described antibody is combined with biomarker, antitumor drug, toxin, radioactivity agent, wherein said biomarker is vitamin H, horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, phycoerythrobilin, Cy3, Cy5 or nanometer magnetic bead, described antitumor drug is carboplatin, cis-platinum or 5-Fluorouracil, toxin is Ricin or lymphotoxin, and the radioactivity agent is 131Iodine, 90Yttrium or radioactivity copper.
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JP6305919B2 (en) * 2011-06-06 2018-04-04 プロセナ バイオサイエンシーズ リミテッド MCAM antagonists and methods of treatment
CN107929732A (en) * 2011-12-12 2018-04-20 中国科学院生物物理研究所 The application of CD146 and its antibody diagnosis and treatment triple negative breast cancer
CN103275225A (en) * 2013-05-10 2013-09-04 洪建� Anti-CD31/CD146 double-specific monoclonal antibody with high affinity and application thereof
CN104083762A (en) * 2014-06-27 2014-10-08 中国科学院生物物理研究所 Reagent for blocking interaction between Netrin-1 and CD146 and tumor treatment application thereof
JP6751104B2 (en) * 2015-04-21 2020-09-02 ユニヴェルシテ デクス−マルセイユＵｎｉｖｅｒｓｉｔｅ Ｄ’Ａｉｘ−Ｍａｒｓｅｉｌｌｅ Use of soluble CD146 as a biomarker for selecting in vitro fertilized embryos for implantation in mammals
CN106589124B (en) * 2016-11-29 2019-03-15 中国人民解放军陆军军医大学第一附属医院 CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell
CN110967489B (en) * 2018-09-28 2021-09-21 中国科学院生物物理研究所 Application of soluble CD146 as blood brain barrier damage marker in central nervous system diseases
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