CN1676531A - Recombinant immunotoxin based on pseudomonas exotoxin containing (Arg) 9 peptide segment - Google Patents

Abstract

This invention belongs to anti-carcinoe mbryonic antigen recombination immuno toxin. It is concretely relates to said antigen immuno toxic with (Arg)9 based on pseudomonic extotoxin. Nucleuotide sequence code-connects ectotoxin and the antigen connecting peptide, contains the expressing carrier of the sequence and the xeno-cell of the sequence and the utilization of the prevention and cure of tumor.

Description

A kind of containing (Arg) 9The recombinant immunotoxin based on Pseudomonas exotoxin of peptide section

Technical field

The invention belongs to a kind of recombinant immunotoxin of anticancer embryonal antigen.Specifically, relate to a kind of 9 arginine peptide sections (Arg) that contain ₉The recombinant immunotoxin based on the anticancer embryonal antigen of Pseudomonas exotoxin, coding connects the nucleotide sequence of extracellular toxin and anticancer embryonal antigen connection peptides, contains the expression vector and host cell that contains this expression vector and the application in preparation medicine for treating tumor thing of described sequence.

Background technology

The toxin that some bacterium and plant produce links to each other with the molecule of somatomedin, antibody or other tool targeted cells effect and can be used for selectivity and kill and carry corresponding acceptor or antigenic target cell (Pastan etc., 1986; Vitetta etc., 1987; ).(pseudomonas exotoxin A is a kind of high active monomeric protein that has PE) to ETA in this toxoid, is that the immunotoxin of fundamental construction has tempting application prospect with PE.In order to the PE molecule that makes up immunotoxin various ways is arranged, comprise that PE40, PE38, PE37, PE35 and corresponding C end are the improved form of KDEL (pastan etc., 1997).The prerequisite of immunotoxin performance cell killing effect is the internalization (internalization) of immunotoxin.Some antibody is because the receptor mediated endocytosis (receptor-mediatedendocytosis) after can not causing the antibody antigen combination effectively, therefore the immunotoxin activity that makes up is not high, perhaps even can not be as candidate's antibody of immunotoxin, thereby the range of application that makes immunotoxin is subjected to certain restriction (Matthew etc., 2000).

PE is a single chain molecule of being made up of 613 amino acid Pseudomonas aeruginosa (Pseudomonas areuginosa) excretory, is made up of three structural domain Domain I, Domain II, Domain III, and wherein Domain I is divided into a, b two portions.Ia (1-252) is a binding domains, combines with the alpha2 Macroglobulin receptor-specific on eukaryotic cell surface; II (253-364) is a transposition structural domain, crosses over endoplasmic reticulum at toxin and enters in the process of kytoplasm and play a role; III (400-613) is a catalyst structure domain, comprises the ADP ribosylation enzyme, at NAD ⁺Acting in conjunction under cause elongation factor 2 (EF2) ribosylation in the eukaryotic cell and inactivation causes that the intracellular protein synthetic suppresses, and inspires apoptotic generation, thereby causes the death of cell.Ib (365-399) separates II and two structural domains of III, and function is not clear.5 amino acid REDLK of the C-terminal 609-613 of PE molecule are that the PE molecule enters behind the cell with kdel receptor in the golgi body and combines essential sequence, make the PE can be effectively by anti-phase endoplasmic reticulum (Hwang etc., 1987 of entering of golgi body; Allured etc., 1986).PE38 is with a kind of clipped form after 365-380 amino acid removes among the Ia of PE molecule and the Ib.PE38KDEL changes the REDLK sequence of natural PE molecule C-terminal into the KDEL sequence on the basis of PE38, thereby the PE molecule is more effectively combined with kdel receptor, thereby improves its biologic activity (Brinkmann etc., 1991).PE35/KDEL clips 253 ~ 279 and 365 ~ 380 aminoacid sequence on the basis of PE38/KDEL, removed the disulfide linkage in the toxin, make toxin after entering cell, need not and directly bring into play catalytic activity through the effect of intracellular protein enzymolysis, make active (Mansfield etc., 1996) (Fig. 1 .A) of raising.

The prerequisite that the immunotoxin performance kills and wounds the target cell biological action is that immunotoxin can effectively and apace enter in the cell, i.e. the internalization of immunotoxin.Usually the internalization of immunotoxin is to be caused by the receptor mediated endocytosis that causes after part and the receptors bind.The internalization of antibody is subjected to influence of various factors: acceptor type; In conjunction with epi-position; Internalization efficient; The release of medicine in the endocytosis corpusculum; Antigen with effectively be anchored on the film again after antibody separates.The bound energy of some part and acceptor promotes its internalization effectively, EGF acceptor for example, ErbB2 acceptor, Transferrins,iron complexes (transferrin) acceptor etc.; The combination of some antigen-antibody also can cause the internalization of antibody, but speed is slower, nerve cell adhesion molecule (NCAM) for example, the membrane antigen of prostate specific (PSMA), carcinomebryonic antigen (CEA) and Saliva Orthana etc.; And most antibody antigen is in conjunction with not causing effective antibody internalization (Ulrik etc., 2000).This just makes the range of application of immunotoxin be restricted greatly.Comparatively successfully be applied at present the clinical immunotoxin overwhelming majority concentrate on several classes can be fast effectively on the acceptor of internalization.(Carcinoembryonic antigen is that nineteen sixty-five Gold and Freedman at first find from fetus and colon cancer tissue CEA) to carcinomebryonic antigen.CEA belongs to non-organ specificity tumor associated antigen, and the tumour of secretion CEA is positioned at hollow organ mostly, as gi tract, respiratory tract, urinary tract etc.Therefore, can be used for targeted therapy at the immunotoxin of CEA to kinds of tumors.Yet, make the biological activity of anti-CEA immunotoxin can not reach higher level, thereby limited its clinical application because CEA is a kind of internalization speed antigen slowly.

Some albumen comprises certain makes albumen enter intracellular active transposition subunit by plasma membrane.Typical example is exactly the Tat in the proteic basic structure of HIV-1 territory _48-67(RKKRRQRRR) subunit (Lindgren etc., 2000 of aminoacid sequence formation; Jeang etc., 1999).In order to set up the substructure of the required optimization of transmembrane transport, exploitation can be transported to each quasi-molecule intracellular transmission molecule, and investigators have made up a series of Tat _48-67Similar molecule.Their transmembrane transport function has obtained confirmation in the Jurkat cell.By a series of similar molecular studies are shown Tat _48-67In the main effect of performance in striding the film transportation of positively charged amino-acid residue.It is not enough that yet charge effect is only arranged, and is positively charged amino-acid residue equally, the peptide section that Histidine, Methionin and ornithine constitute, and the efficient of its transmembrane transport is significantly less than Tat _48-67On the contrary, a kind of film conveying efficiency of striding of the peptide section (R9) that is made of 9 L-type arginine residues is Tat _48-6720 times; The film conveying efficiency of striding of the peptide section (r9) that is made of 9 D-type arginine residues is Tat _48-67More than 100 times (Wender etc., 2000).These studies show that Tat _48-67In the arginine residues component in the transmembrane transport function, bringing into play than charge effect and the even more important effect of amino acid backbone structure.Therefore, the abundant peptide section of arginine can be used for all kinds of molecule transport to cell interior.

Above background technology relates to relevant quoted passage, and the reference after the specific embodiments of the present invention is seen in its detailed source.

Summary of the invention

Described based on background technology, most antibody antigen is in conjunction with not causing effective antibody internalization.CEA is a kind of internalization speed antigen slowly, makes the biological activity of anti-CEA immunotoxin can not reach higher level, thereby has limited its clinical application, and this just makes the range of application of immunotoxin be restricted greatly.Some antibody is because the receptor mediated endocytosis after can not causing the antibody antigen combination effectively, therefore the immunotoxin activity that makes up is not high, perhaps even can not be, as candidate's antibody of immunotoxin thus also make the application of immunotoxin be subjected to certain restriction.

The problems referred to above at present existence, the object of the present invention is to provide a kind of Pseudomonas exotoxin PE35/KDEL to constitute by anti-CEA single-chain antibody and brachymemma and transformation, and between 607 amino acids of toxin and single-chain antibody, add different connection peptides, can be transported to fast and effectively in the cell by the anti-CEA immunotoxin that internalization is more weak, significantly improve a kind of novel anti CEA immunotoxin that it kills and wounds the target cell effect.The single chain antibody fragments gene of anti-CEA is VH and the VL partial amino-acid series by the anti-CEA monoclonal antibody of bibliographical information (reference 11), with repeated connection peptides (G ₄S) ₃Connect, reverse translation becomes nucleotide sequence, synthesizes, is spliced through this laboratory.

The present invention provides aminoacid sequence and its amino acid whose nucleotide sequence of this connection peptides of encoding of the connection peptides between described anti-CEA single-chain antibody and the toxin particularly.

The connection peptides aminoacid sequence is as follows respectively:

(Gly ₄Ser) ₂Connection peptides: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser; (Arg) ₉(Gly ₄Ser) ₂Connection peptides: Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly GlyGly Gly Ser Gly Gly Gly Gly Ser.

The nucleotide sequence of described coding connection peptides is not limited to but comprising nucleotide sequence as follows:

Coding (Gly ₄Ser) ₂The nucleotide sequence of connection peptides:

GGC?GGC?GGC?GGC?ACG?GGT?GGT?GGT?GGT?AGC

Coding (Arg) ₉(Gly ₄Ser) ₂The nucleotide sequence of connection peptides:

CGC?CGC?CGT?CGT?CGC?CGC?CGC?CGC?CGT?GGC?GGC?GGC?GGC?ACG?GGT?GGT?GGTGGT?AGC

Another object of the present invention provides a kind of expression vector that contains aforesaid nucleotide sequence, and they are respectively as shown in Figure 2 expression vector pCIT35g, pCIT35a.

Another object of the present invention provides a kind of host cell aforesaid nucleotide sequence or pCIT35g or pCIT35a expression vector that contains.One of them, host cell is selected from intestinal bacteria E coli.

A further object of the invention provides a kind of medicine that is used for the treatment of tumour, one of them, aforesaid anti-CEA immunotoxin, provide a kind of in other words conj.or perhaps according to relevant technologies step of the present invention, in the method for preparing the medicine for treating tumor thing, this method can be treated the tumor associated antigen medicine in preparation, as: use in nasopharyngeal carcinoma and the colorectal carcinoma medicine.

In addition, on the basis of the application's contextual disclosure, others of the present invention are conspicuous to those skilled in the art.

Involved in the present invention with Pseudomonas exotoxin immunotoxin A (Pseudomonas exotoxinA, clipped form PE35 PE) is the recombinant immunotoxin of fundamental construction, its PE35 is a kind of clipped form of PE.On the basis of natural PE, the aminoacid sequence of 253～279 among binding domains Domain Ia (1～252) and the transposition structural domain Domain II (253 ~ 364) is clipped, removed the disulfide linkage in the toxin, make immunotoxin enter the process that need not to pass through proteasome degradation behind the cell, play a role and directly enter in the cell with activity form.PE35/KDEL is the form that the C-terminal aminoacid sequence of PE35 is sported KDEL by REDLK, can improve the efficient of the receptors bind on toxin and the golgi body, make toxin can more effectively oppositely enter into endoplasmic reticulum, and then be transported to and bring into play biological action (Fig. 1 .A) in the kytoplasm.Be the immunotoxin of fundamental construction with PE35 owing to need not process, its activity is improved greatly than corresponding PE38 immunotoxin through proteolytic cleavage.In the PE35 immunotoxin, antibody molecule normally links to each other with toxin with chemical coupling (idol) connection and two kinds of forms of gene recombination.Form for the many forms structures of the immunotoxin that makes up with single-chain antibody (scFv) with gene recombination.In the PE35 immunotoxin, single-chain antibody gene is usually located at the downstream of 607 amino acids of toxin, is right after 604～613 aminoacid sequence again, and the insertion of this form is proved to be the performance (Fig. 1 .B) that does not influence the toxin function.

The antibody molecule that is used to make up immunotoxin mostly with corresponding antigen or receptors bind after cause fast and effectively receptor mediated endocytosis (Receptor-mediatedendocytosis), this also is the prerequisite of immunotoxin performance biological function.Can not be effectively and the antibody of internalization fast for great majority, the activity of immunotoxin will be had a greatly reduced quality.(Carcinoembryonic antigen CEA) is a kind of internalization speed ratio antigen more slowly to carcinomebryonic antigen.A kind of anti-CEA PE35 immunotoxin that the present invention makes up is a gene fragment of directly inserting anti-CEA single-chain antibody in the gene downstream of 607 amino acids of toxin-encoding, is right after the gene of coding 604～613 amino acids sequences again.The immunotoxin of this form is compared with corresponding PE38 immunotoxin, and activity there is no raising, and slightly descends.Reason mainly is that antibody sequence is positioned at toxin inside, and antigen-binding activity is affected, and internalization reduces, and causes active decline.Between antibody and toxin 607 amino acids, add connection peptides (Gly ₄Ser) ₂After, the improving of antibody in conjunction with activity, killing activity and the difference of PE38 immunotoxin are little.It is not obvious to illustrate that anti-CEA antibody combines the internalization that causes the back with antigen.

It is provided by the present invention that a kind of (Pseudomonasexotoxin A, clipped form PE35 PE) is the recombinant immunotoxin of fundamental construction, is to insert (Arg) between 607 amino acids of toxin and antibody sequence with Pseudomonas exotoxin immunotoxin A ₉(Gly ₄Ser) ₂Both kept antigen-binding activity and specificity unaffected, significantly improved the internalization efficient of immunotoxin again, the cell killing activity of immunotoxin is significantly improved, the present invention with corresponding without the PE35 immunotoxin of transforming mutually specific activity improve greatly, have significant technical progress.The present invention can be applicable to self the effectively antibody that is considered to be not suitable for being configured to immunotoxin of internalization, but the range of application of immunotoxin is improved greatly, has good practicability.

At present, do not see the report identical both at home and abroad with the present invention.

Description of drawings

The PE that accompanying drawing 1. is multi-form and be the structural representation of the anti-CEA immunotoxin of fundamental construction with PE

1.A. Pseudomonas exotoxin and block the structural representation of forms of modification

(a)PE；(b)PE38/KDEL；(c)PE35/KDEL

1.B. with PE38/KDEL and PE35/KDEL is the structural representation of the anti-CEA immunotoxin of fundamental construction

On: CEA (Fv)/PE38/KDEL; Down: PE35/CEA (Fv)/KDEL

The structural representation of accompanying drawing 2. plasmid pCIT35, pCIT35g, pCIT35a

The SDS-PAGE of accompanying drawing 3. expression products and western blotting analyze

The SDS-PAGE of Fig. 3 .A. expression product analyzes

A：PE35/GS/CEA(Fv)/KDEL；B：PE35/r9/CEA(Fv)/KDEL

The SDS-PAGE of Fig. 3 .B. expression product inclusion body washing analyzes

A: molecular weight of albumen standard; B: the ultrasonic supernatant of expression product;

C: expression product ultrasound precipitation; D: the inclusion body after the washing

The western blotting of Fig. 3 .C. expression product analyzes

a：PE35/GS/CEA(Fv)/KDEL；b：PE35/r9/CEA(Fv)/KDEL

C: dye the molecular weight of albumen standard in advance

Accompanying drawing 4. flow cytometries are measured cell in conjunction with activity

The burnt micro-image of the copolymerization of the anti-CEA immunotoxin internalization that accompanying drawing 5. is multi-form

A：CEA(Fv)/PE38/KDEL B：PE35/CEA(Fv)/KDEL

C：PE35/GS/CEA(Fv)/KDEL D：PE35/r9/CEA(Fv)/KDEL

The burnt micro-image of copolymerization of the internalization effect of PE35/r9/CEA (Fv)/KDEL under 6. different action times of accompanying drawing and the different operative temperature; A:37 ℃ of 0.5hr, B:37 ℃ 1h, C:37 ℃ 2h,

A D:4 ℃ of 2h left side: FITC dyeing; In: PI dyeing; Right: superimposed image

The multi-form anti-CEA PE35 immunotoxin of accompanying drawing 7. is to the lethal effect of KB cell CNE-2

Accompanying drawing 8.PE35/r9/CEA (Fv)/KDEL is to the lethal effect of different tumour cells.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.

The synthetic suitably nucleotide fragments of size of design obtains (Arg) by overlapping pcr amplification ₉(Gly ₄Ser) ₂Gene order, and introduce suitable restriction enzyme site endways.Amplified production behind digestion with restriction enzyme, is inserted among PE35/CEA (the Fv)/KDEL expression vector pCIT35 after corresponding enzyme is cut, and structure contains connection peptides (Gly ₄Ser) ₂Immunotoxin PE35/GS/CEA (Fv)/KDEL expression vector pCIT35g and contain connection peptides (Arg) ₉(Gly ₄Ser) ₂The expression vector pCIT35a of immunotoxin PE35/r9/CEA (Fv)/KDEL.Plasmid transformed host cell coli strain BL21 (DE3) star with expression vector pCIT35a that builds and pCIT35g.Picking positive colony bacterium is transferred in the LB substratum that contains penbritin 100 μ g/ml, and 37 ℃ are cultured to OD ₅₅₀Be about 0.5, adding final concentration in culture is the IPTG of 0.4mmol/l, inducing culture 4 hours.Collect expression product, the ultrasonication thalline, the centrifugal 10min of 12000rpm goes up cleer and peaceful precipitation and does not carry out 12%SDS-PAGE analysis and western engram analysis.The inclusion body of expressing is through washing process, and sex change is after the method renaturation that dilution and the dialysis of denaturing agent concentration gradient combine.Product carries out the evaluation of biologic activity, comprises flow cytometry identification of cell surface antigen in conjunction with activity, and the burnt microtechnique of indirect immunofluorescence copolymerization is identified the internalization character of product, tetrazolium salts (MTT) method identification of cell killing activity.The concrete operations flow process is as follows:

The structure of embodiment 1, expression vector pCIT35:

PE35KDEL is a kind of brachymemma and the forms of modification of PE molecule, its gene fragment is that gene with PE38 is (from carrier pMS8-38f+T, be so kind as to give by NIH doctor Brinkmann) for template, design following primer: (1) Nde35:5 '-gACATATgTgggAACAACTggAgCAgTgC-3 ' and

(2) EcoR:5 '-CAggAATTCATATTCgATTgggCTg-3 ' amplification transformation obtains.

After the nucleotide sequence site of coding 607 amino acids of PE35KDEL,, insert the gene fragment of anti-CEA single-chain antibody, obtain the gene fragment of PE35/CEA (Fv) KDEL with the restriction enzyme site of XhoI and SacI.PCIT35 is inserted into NdeI with the gene fragment of PE35/CEA (Fv)/KDEL on the basis of universal expression vector pET21a (+) (Novagen company), make up in the EcoRI double enzyme site and form (Fig. 2 .A).

The structure of embodiment 2, expression vector pCIT35a:

This expression vector will contain (Gly on the basis of pCIT35 ₄Ser) ₂The primer of connection peptides nucleotide sequence is that template amplification goes out to contain (Gly with the pCIT35 plasmid ₄Ser) ₂And 5 ' end contains SacII,, 3 ' end contains the nucleotide fragments of XhoI, inserts in the pCIT35 carrier of same restrictions restriction endonuclease digestion behind the double digestion, is built into expression vector pCIT35g (Fig. 2 .B).To contain (Arg) ₉(Gly ₄Ser) ₂The primer of connection peptides nucleotide sequence is that template amplification goes out to contain (Arg) with the pCIT35 plasmid ₉(Gly ₄Ser) ₂And 5 ' end contains SacII, and 3 ' end contains the nucleotide fragments of XhoI, inserts in the pCIT35 carrier of same restrictions restriction endonuclease digestion behind the double digestion, is built into expression vector pCIT35a (Fig. 2 .C).

Synthetic 4 oligonucleotide fragments of design,

SacFor：5’-CAC CCgCggCACgCAgAACT-3’ 20nts

GSArg1：5’- ACGACGGCGGCG ATCGAT

CGGTTTGCCGGG-3’ 48nts

GSArg2：5’-CCG CTCGAGGCTACCACCACCACCCGTGCCGCCGCC

-3’ 54nts

GS：5’-CCG CTCGAGGCTACCACCACCACCCGTGCCGCCGCCGCCA

ATCGATCGGTTTGCCGGG-3’ 58nts

Underscore mark restriction enzyme site; Dash area is a complementary nucleotide sequence among the overlapping PCR.

1. the extraction of carrier pCIT35 plasmid

Take alkaline process to extract plasmid in a small amount: single bacterium colony of the positive colony behind the picking pCIT35 transformed into escherichia coli host bacterium TOP10 is 37 ℃ of overnight incubation in 5ml LB nutrient solution (containing Amp 100 μ g/ml), and 12000rpm collected thalline in centrifugal 1 minute.Precipitation is suspended among the 100 μ lSolutionI (50mmol/l Glucose, 10mmol/l EDTA, 25mmol/l Tris pH8.0), fully mixing.Add the new preparation of 200 μ l SolutionII (0.2mol/l NaOH, 1%SDS), the tight pipe lid of lid, soft puts upside down 4-5 time, with centrifuge tube as for placement in the ice bath 3 minutes.(3mol/l Kac pH4.8), puts upside down for several times repeatedly, places 5 minutes in ice bath to add the SolutionIII of 150 μ l precoolings.4 ℃, centrifugal 10 minutes of 12000rpm changes supernatant in another centrifuge tube over to.The phenol that adds equivalent volumes: chloroform (1: 1), vibration mixes organic phase and water, in 4 ℃ with 12000rpm centrifugal 2 minutes, supernatant is transferred in another centrifuge tube.In precipitation at room temperature nucleic acid, vibration mixes with 2 times of volume of ethanol, places 2 minutes in room temperature.In 4 ℃, centrifugal 5 minutes of 12000rpm collects the nucleic acid precipitation.The careful suction removed supernatant liquor, and centrifuge tube is inverted on the paper handkerchief, drains so that all liquid flows out.Add 1ml70% ethanol in precipitation, washing DNA precipitation is dissolved in 50 μ lddH after the precipitation drying ₂Among the O.

2. overlapping pcr amplification contains the nucleotide fragments of connection peptides sequence

2.1 coding contains Arg ₉(Gly ₄Ser) ₂The amplification of the nucleotide sequence of connection peptides

With the pCIT35 plasmid is that template takes the amplification of two steps to obtain full length fragment.The first step is that each 10pmol of upstream and downstream primer adds in the centrifuge tube with SacFor and GSArg1, adds plasmid template 1 μ l, 10 * PCR buffer, 2 μ l, and 2 μ ldNTPs (2mmol/l), 0.2 μ l pfu archaeal dna polymerase (5u/ μ l) is used ddH ₂O supplies cumulative volume to 20 μ l, and mixing carries out 30 and takes turns PCR circulation: 94 ℃ of sex change 1 minute, and 55 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds.The PCR product carries out 1% agarose gel electrophoresis, utilizes the Shen to reclaim Kit by lottery industry sepharose DNA, reclaims required dna fragmentation.With the dna fragmentation that reclaims is that template adds 10pmol in centrifuge tube, adds primer SacFor and each 10pmol of GSArg2,10 * PCR buffer, 2 μ l, and 2 μ ldNTPs (2mmol/l), 0.2 μ lpfu archaeal dna polymerase (5u/ μ l) is used ddH ₂O supplies cumulative volume to 20 μ l, and mixing carries out 30 and takes turns PCR circulation: 94 ℃ of sex change 1 minute, and 30 seconds ℃ of 55 ℃ of annealing, 72 ℃ were extended 40 seconds.The PCR product carries out 1% agarose gel electrophoresis, utilizes the Shen to reclaim Kit by lottery industry sepharose DNA, reclaims required dna fragmentation.

2.2 coding contains (Gly ₄Ser) ₂The amplification of the nucleotide sequence of connection peptides

With the pCIT35 plasmid is that template takes the amplification of two steps to obtain full length fragment.The first step is that each 10pmol of upstream and downstream primer adds in the centrifuge tube with SacFor and GS, adds plasmid template 1 μ l, 10 * PCR buffer, 2 μ l, and 2 μ ldNTPs (2mmol/l), 0.2 μ l pfu archaeal dna polymerase (5u/ μ l) is used ddH ₂O supplies cumulative volume to 20 μ l, and mixing carries out 30 and takes turns PCR circulation: 94 ℃ of sex change 1 minute, and 55 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds.The PCR product carries out 1% agarose gel electrophoresis, utilizes the Shen to reclaim Kit by lottery industry sepharose DNA, reclaims required dna fragmentation.

3.PCR the digestion with restriction enzyme of amplified production and purifying

With SacII and XhoI pcr amplification product is digested: get about 1 μ g dna fragmentation (1 * buffer K in 40 μ l reaction systems, the SacII of 30u and the XhoI of 30u), 37 ℃ of enzymolysis 4 hours, after 1% agarose gel electrophoresis detects digestion fully, reclaim required band at long-wave ultra violet lamp incision glue, can reclaim the kit explanation by lottery industry biotech company glue with reference to the Shen, reclaim and the required endonuclease bamhi of purifying with post.

4. the digestion with restriction enzyme of vector plasmid pCIT35 and purifying

With SacII and XhoI carrier DNA is digested: get 1 μ g plasmid (1 * Buffer K in 40 μ l reaction systems, the SacII of 30u and the XhoI of 30u), 37 ℃ of enzymolysis 4 hours, after 1% agarose gel electrophoresis detects digestion fully, reclaim required band at long-wave ultra violet lamp incision glue, can reclaim the kit explanation by lottery industry biotech company glue with reference to the Shen, reclaim and the required endonuclease bamhi of purifying with post.

5.DNA ligation

Fetch the about 40ng of double digestion pCIT35 of receipts, double digestion PCR fragment 20ng adds 2 μ l10 * T ₄Ligase enzyme (about 20u) adds ddH ₂O is to cumulative volume 20 μ l, mixing, and 16 ℃ of connections are spent the night.

6. the preparation of competent cell

Prepare colibacillary competent cell with calcium chloride: picking mono-clonal from the culture plate, switching 37 ℃ of shaking culture in the 5ml substratum are spent the night.Be forwarded in 20 ~ 40ml antibiotic-free substratum in 1: 100 ratio, quick oscillation is cultured to OD ₆₀₀Be 0.4 ~ 0.6 o'clock, ice bath 15 minutes.4 ℃ of 4000rpm collected thalline in centrifugal 10 minutes, abandoned supernatant, added the 0.1mol/LCaCl of 20ml precooling ₂, resuspended mixing, ice bath 40 minutes.Centrifugal 10 minutes of 4 ℃ of 4000rpm abandon the 15% glycerine 0.1mol/L CaCl that contains that adds precooling behind the supernatant ₂1 ~ 2ml has hanged thalline, packing gently.Be stored in-80 ℃.

7. the conversion of recombinant DNA, the screening of positive colony and order-checking are identified

In 200 μ l competent cells, add 10 μ l and connect mixture, mixing.Ice bath 30 minutes, 42 ℃ of heat shocks 100 seconds, ice bath 2 minutes rapidly then.Add 0.8ml LB substratum, and 37 ℃ of jogs (＜150rpm) recovery in 45 minutes, 10, centrifugal 1 minute of 000rpm abandons supernatant, adds the resuspended precipitation of 100～200 μ lLB substratum, coats on the LB flat board that contains Amp 100 μ g/ml 37 ℃ of overnight incubation.Single bacterium colony of growing on the picking flat board adopts alkaline process to extract plasmid in a small amount, uses SacII and XhoI double digestion respectively; SacFor, GSArg2 are that the PCR of primer identifies the segmental plasmid of insertion external source.Send the order-checking of Bo Ya biotech company to identify the plasmid after identifying, identify the correct positive plasmid pCIT35a of back order-checking.

Embodiment 3, contain the expression of the anti-CEA immunotoxin of different connection peptides

1. the expression of immunotoxin

Expression vector plasmid pCIT35a and pCIT35g transformed into escherichia coli E.coli bacterial strain BL21 (DE3) star, (Novagen company) picking mono-clonal is 30 ℃ of overnight incubation in LB (containing 100 μ g/ml Amp) substratum.With 10% ratio switching, 37 ℃ grow to OD ₆₀₀About 0.6-0.8, adding IPTG is 0.4mmol/ml to final concentration, continuation was cultivated 4 hours, centrifugal collection thalline.Thalline is resuspended in the ultrasonic damping fluid, and freeze thawing three times is centrifugal, 12000rpm, 30 minutes.Precipitation is resuspended in the ultrasonic damping fluid ice-bath ultrasonic.Cleer and peaceful precipitation in the centrifugal collection is carried out 12%SDS-PAGE and is analyzed.

The preparation of polyacrylamide sees table 1:

Concentrate glue 5 separation gels 12 sealing compounds

％ ％

30％Acr/Bis(29∶1)(ml)?0.5 6.0 0.53

5M?Tris-HCl(pH8.8)(ml)?- 3.8 -

1.0M 0.38 - 0.5

Tris-HCl(pH6.8)(ml) 0.03 0.15 0.02

10％SDS(ml) 0.03 0.15 0.02

10％AP(ml) 0.003 0.006 0.0012

TEMED(ml) 2.1 4.9 0.93

ddH ₂O

The processing of sample: an amount of protein sample and isopyknic 2 * sds gel sample loading buffer (100mmol/Tris-HCl pH6.8,200mmol/DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine) mix, and sample is treated in boiling water bath heating 5 minutes.

Electrophoresis: electrophoretic buffer 25mmol/Tris-HCl, pH8.3,0.1%SDS.Get sample on the sample of an amount of volume, constant voltage 60V treats that sample enters separation gel, constant voltage 120V.

Dyeing and decolouring: (the 0.25g coomassie brilliant blue R250 is dissolved in (90ml methyl alcohol: water (1: 1, v/v) with the 10ml glacial acetic acid), room temperature dyeing 6-12 hour in 100ml methyl alcohol-glacial acetic acid solution to adopt coomassie brilliant blue R250 dyeing.With methyl alcohol-glacial acetic acid solution decolouring, the room temperature decolouring is taken a picture to having obvious band, and the result is referring to accompanying drawing 3.A, and two kinds of immunotoxins have obvious protein band about the 66kDa of molecular weight standard.

Western trace: the isolating sample of the above-mentioned SDS-PAGE Hoefer TE 22 Mighty Small of amersham pharmacia biotech ^TMTransphor Tank Transfer Unit device, at transfering buffering liquid (39mmol/ glycine, 40mmol/l Tris alkali, 0.03%SDS, 20% methyl alcohol) 400mA shifted 1.5 hours, and the film after the transfer printing is put into the confining liquid sealing, and (confining liquid: 5% skimmed milk was dissolved in TBS (TBS:100mmol/l Tris-HCl in 2 hours, pH7.5,0.9%NaCl), give a baby a bath on the third day after its birth time each 5 minutes with TBST (TBS that contains 0.1%Tween 20).Combine 1 hour with the anti-E-tag mouse monoclonal antibody room temperature that was diluted among the TBS by 1: 1000, TBST gives a baby a bath on the third day after its birth inferior.In conjunction with 1 hour, TBST washed 5 times by the sheep anti-mouse antibody room temperature of the HRP mark of 1: 5000 dilution proportion in TBS in adding again.(6mg/ml DAB, 1%H in substrate colour developing liquid at last ₂O ₂) developed the color 5-15 minute, take pictures, the results are shown in accompanying drawing 3.C.

2. the renaturation of inclusion body

Inclusion body is through washing (50mM Tris-HC, pH8.0; 50mM Tris-HC, pH8.0,10mMEDTA and 2%Triton each once, each 30 minutes) after, purity can obviously improve (Fig. 3 .B).Inclusion body protein after the washing is dissolved in the inclusion body lysate (10mM pH7.2 sodium phosphate buffer, 8M urea), 37 ℃ of cracking 1 hour, and 12, centrifugal 30 minutes of 000rpm gets supernatant.Metaprotein carries out dialysis the 10mM pH7.2 sodium phosphate buffer that contains 4M urea, and with 10 times of dilution refolding liquid dilutions, 4 ℃ of placements are spent the night and made protein renaturation then.The protein solution of renaturation pack into dialysis tubing to PBS solution dialysed overnight, 12, centrifugal 20 minutes of 000rpm gets supernatant, measures protein concentration, packing, 4 ℃ of storages are standby.

The determination of activity of embodiment 4, immunotoxin

3. the immunotoxin cell is in conjunction with active mensuration

Adopt the method for indirect fluorescent mark, the anti-CEA immunotoxin of utilization Flow cytometry is active to the combination of target cell.10 ⁵Individual KB cell CNE-2 is hatched 90min with the immunotoxin 200 μ l of 10 μ g/ml in 4 ℃ respectively, and the cell that does not add sample compares group, and PBA (1%BSA/PBS) gives a baby a bath on the third day after its birth time.Add by the mouse-anti E-tag monoclonal antibody 200 μ l after the dilution in 1: 1000 and hatch 60min for 4 ℃, PBA gives a baby a bath on the third day after its birth inferior.Add 200 μ l FITC-sheep anti mouse Ig (1 μ g/10 ⁶Cell) hatch 60min for 4 ℃, PBA gives a baby a bath on the third day after its birth inferior, and flow cytometer detects.Fig. 4 result shows, adds (Gly between antibody and toxin ₄Ser) ₂The cell that can effectively improve immunotoxin is in conjunction with activity, avoids the reduction of the antigen binding capacity that the change because of the antibody on position causes.

4. the internalization of immunotoxin experiment

In the hole of 6 porocyte culture plates, place cover glass, by every hole 10 ⁵Individual cell is laid on the CNE-2 cell in the hole, in 37 ℃, and 5%CO ₂Incubated overnight.Confide all nutrient solution, in the hole, add 10 μ g/ml immunotoxin sample 1ml respectively, set up control wells, add the PBA of equal volume.In 37 ℃ of incubation 2h.After PBA gives a baby a bath on the third day after its birth time, give a baby a bath on the third day after its birth time with the Gly-HCl damping fluid of pH4.0,10min at every turn is to remove cell-surface antigens bonded immunotoxin.After PBA gave a baby a bath on the third day after its birth time, in the fixing 5min of room temperature, PBA gave a baby a bath on the third day after its birth inferior with 4% Paraformaldehyde 96.Add ice acetone effect 30s, PBA gives a baby a bath on the third day after its birth inferior.Add by 1: 1000 dilution back 9E10 ascites 1ml and hatch 60min for 4 ℃, PBA gives a baby a bath on the third day after its birth inferior.Add 1ml and hatch 60min for 4 ℃ by the FITC-sheep anti mouse Ig of dilution in 1: 500, PBA gives a baby a bath on the third day after its birth inferior.Cover glass is taken out from culture plate, and back-off has on the slide glass of mountant (90% glycerine, 10%PBS, 5mM Ursol D) in dropping, observes under Laser Scanning Confocal Microscope.The result shows, inserts Arg between antibody and toxin ₉The peptide section can effectively improve the internalization efficient of immunotoxin, and at short time and 4 ℃ all can effectively play a role (Fig. 5,6).

3. the external killing activity of immunotoxin is measured

The tumour cell CNE-2, the SW1116 that dilute well-grown expression CEA reach Proliferation of Human Ovarian Cell SKOV3 and the KB cell CNE-1 10 that does not express CEA ⁵Individual/ml, every hole 100 μ L spread 96 porocyte culture plates, put 5%CO ₂Constant incubator is cultivated.When being paved with the hole basal surface, add the immunotoxin of different concns at cell, continue to cultivate 48h.Adding final concentration is 0.5mg/ml tetrazolium salts (MTT) solution incubation 4h, confides all nutrient solution.Be attached to the oxidation state MTT purple crystal of cultivating on the hole wall and fully dissolve with dimethyl sulfoxide (DMSO) (DMSO), 570nm measures optical density value.Every kind of cell is all established dead cell and viable cell contrast, and other step is the same.The tumor cytotoxicity effect is calculated by following formula:

Fig. 7,8 result show, contain Arg ₉Anti-CEA/PE35 immunotoxin obviously improve the specific killing of tumour cell is active.

Reference

1.Pastan I, Willingham MC ， ﹠amp; FitzGerald DJ: immunotoxin (Immunotoxins). cell (Cell), 1986,47 (5): 641-648.

2.Vitetta ES, Fulton RJ, May RD, etc.: the meter of reseting of natural toxin is used to make up anti-tumor agent (Redesigning nature s poisons to create anti-tumorreagents). science (Science), 1987,238 (4830): 1098-1104.

3.Hwang J, FitzGerald DJ, Adhya S, etc.: the functional domain (Function domains ofPseudomonas exotoxin identified by deletion analysis of the geneexpressed in E.coli) of identifying Pseudomonas exotoxin by the expression of missing gene in intestinal bacteria. cell (Cell); 1987,48:129-136.4.Allured VS, Collier RJ, Carroll SF, Deng: the structure of the ETA of 3.0 dust resolving power (Structure of exotoxin A of Pseudomonas aeruginosa at 3.0 Angstromresolution). PNAS (Proc Natl Acad Sci USA), 1986,83:1320-1324.

5.Brinkmann U, Pai LH, FitzGerald DJ, Deng: B3 (Fv)-PE38KDEL, a kind of monochain immunotoxin that people's tumour disappears fully in the mouse (B3 (Fv)-PE38KDEL that makes, asingle-chain immunotoxin that causes complete regression of a humancarcinoma in mice). PNAS (Proc Natl Acad Sci USA), 1991,88 (19): 8616-8620.

6.Mansfield E, Pastan I, FitzGerald D J: the character of the immunotoxin RFB4-Pseudomonas exotoxin A of target B cell malignancies cell CD 22 molecule is identified (Characterization of RFB4-Pseudomonas exotoxin A immunotoxinstargeted to CD22 on B cell malignancies.) biological composition chemistry (Bioconjug.Chem), 1996,7 (5): 557 ~ 563.

7.Lindgren, M., H llbrink, M., Prochiantz, A..: cell permeable peptide (Cell-penetrating peptides) .2000, progress in pharmacology (Trends Pharmacol.Sci.) .21,99-103.

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Claims (11)

1. anti-CEA immunotoxin, it is characterized in that being constituting by anti-CEA single-chain antibody and brachymemma and the Pseudomonas exotoxin PE35/KDEL that transforms, and between 607 amino acids of toxin and single-chain antibody, add different connection peptides, can be transported to fast and effectively in the cell by the anti-CEA immunotoxin that internalization is more weak, significantly improve a kind of anti-CEA immunotoxin that it kills and wounds the target cell effect.

2. a kind of anti-CEA immunotoxin according to claim 1, the aminoacid sequence of its different connection peptides, comprising: (Gly ₄Ser) ₂Connection peptides: Gly Gly Gly Gly Ser Gly Gly Gly GlySer; Or (Arg) ₉(Gly ₄Ser) ₂Connection peptides: Arg Arg Arg Arg Arg Arg Arg Arg ArgGly Gly Gly Gly Ser Gly Gly Gly Gly Ser.

3. nucleotide sequence of the described connection peptides of claim 2 of encoding.

4. described (the Gly of claim 2 encodes ₄Ser) ₂The nucleotide sequence of connection peptides:

GGC?GGC?GGC?GGC?ACG?GGT?GGT?GGT?GGT?AGC；

Coding (Arg) ₉(Gly ₄Ser) ₂The nucleotide sequence of connection peptides:

CGC?CGC?CGT?CGT?CGC?CGC?CGC?CGC?CGT?GGC?GGC?GGC?GGC?ACG?GGT?GGT

GGT?GGT?AGC。

5. expression vector that contains with good grounds claim 3 or 4 described nucleotide sequences.

6. expression vector according to claim 5 is a kind of expression vector pCIT35g or pCIT35a as shown in Figure 2.

7. host cell that contains claim 5 or 6 described expression vectors.

8. host cell according to claim 7 is intestinal bacteria E coli.

9. described according to claim 1-8, optional one of them application in preparation medicine for treating tumor thing.

10. the application in preparation nasopharyngeal carcinoma and colorectal carcinoma medicine according to claim 9.

11. require a kind of anti-CEA immunotoxin medicine that is used for the treatment of tumour of preparation according to claim 8 or 9 described claims.

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