CN102321653B - Applications of man-mouse chimeric CD4 (cluster of differentiation 4) plasmid and multiclonal antibody in inhibiting HIV (human immunodeficiency virus) infection - Google Patents
Applications of man-mouse chimeric CD4 (cluster of differentiation 4) plasmid and multiclonal antibody in inhibiting HIV (human immunodeficiency virus) infection Download PDFInfo
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Abstract
The invention discloses a man-mouse chimeric CD4 (cluster of differentiation 4) plasmid, wherein the sequence of the plasmid is showed as SEQ ID No.1. The plasmid preparation method provided by the invention is simple and convenient; the experiment shows that the plasmid constructed by the invention transfects 293T cells with coreceptors, and can effectively mediate HIV virus infection, the inhibition rate of the collected multiclonal antibody of mice after immunization to HIV pseudo viruses of different subtypes after the multiclonal antibody is diluted by 100 times is 60-90%, and favorable broad spectrum neutralization capability is shown.
Description
Technical field
The present invention relates to a kind of mosaic type plasmid, people mouse mosaic type CD4 plasmid specifically, and by this plasmid prepare how anti-.
Background of invention
Over nearly 20 years, HIV-1 vaccine area research result shows, by to HIV-1, the research of SIV and SHIV strongly show cellular immunization that T is cell-mediated and the cell-mediated humoral immunization of B indispensable.Antibody can protect body to avoid virus (as Measles virus, hepatitis virus and influenza virus etc.) infection, still, for the virus of the variation of the height as HIV-1, is difficult to obtain to have in wide spectrum and active antibody by immunity.HIV-1 neutralizing antibody target spot is mainly virus membrane antigen tripolymer and susceptible target cell surface receptor, to stop HIV-1 virus infection target cell.Although HIV-1 by membranin suddenly change, interaction between the covering of glycosylation site, albumen, and the conformation in critical function district the method such as covers and escapes host immune system, but still has 6 wide spectrum neutralizing antibodies to produce by the relative conserved regions of identification HIV-1 membranin.With the variation of HIV-1 membranin height, compare, the characteristic of cell receptor CD4 and accessory receptor high conservative thereof will become the important target spot of therapeutic type antibody in clinical experiment, become the key that HIV-1 enters cell.Have report to show, CCR5 inhibitor maraviroc only stops R5 preferendum HIV-1 target cell infection, and is invalid to X4 and amphitropic virus.For the antibody that CXCR4 is target spot, study more difficult.In recent HIV vaccine research process, research shows that it has the ability of stronger many subtype virus of neutralization strain about the CD4 monoclonal antibody.CD4mAb ibalizumab (being TNX-355 and Hu5A8) is humanization IgG4 monoclonal antibody, in clinical I phase and II phase, testing, shows its antiviral activity.This paper, by building people CD4 and people and mouse mosaic type plasmid DNA immune mouse, by the screening of polyvalent antibody and monoclonal antibody anti-virus ability, obtains to have the monoclonal antibody of wide spectrum neutralising capacity.Hope provides more theory and experiment basis in the future preventative and therapeutic vaccine.
Summary of the invention
The purpose of this invention is to provide a kind of people mouse mosaic type CD4 plasmid.
The second purpose of the present invention is to provide the polyclonal antibody that the HIV pseudovirus is had to obvious inhibition that utilizes that above-mentioned plasmid prepares.
A further object of the present invention is to provide the purposes of described polyclonal antibody.
Invention thinking of the present invention is: the HIV target cell infection is at first by being combined with its surface C D4 acceptor and accessory receptor, if therefore can block the combination of HIV and CD4 acceptor, can effectively suppress HIV infects, we have designed the people mouse mosaic type plasmid for people CD4 acceptor V1V2 zone, by DNA immunization, wish to obtain the having polyclonal antibody that suppresses the HIV function, for exploring HIV vaccine and medicament research and development novel targets, provide theory and experimental basis.
The present invention adopts following concrete technical scheme:
A kind of people mouse mosaic type CD4 plasmid, the sequence of described plasmid is as shown in SEQ ID No.8.
The application of above-mentioned plasmid in preparation mediation HIV virus infective medicament.
The preparation method of above-mentioned people mouse mosaic type CD4 plasmid, described method comprises the steps:
(1) from Ghost cell and mouse boosting cell, extract total RNA respectively;
(2) utilize P1, P2 amplification human total rna to obtain people CD4, the total RNA of P3, P4 amplification mouse obtains mouse CD4;
(3) P3, P10 amplification mouse CD4 obtains mouse CD4 signal peptide; P11, P7 amplification people CD4 obtains people CD4V1V2 region sequence; Utilize P3, P7 amplification mouse CD4 signal peptide and people CD4V1V2 district to obtain mouse CD4 signal peptide and the chimeric product in people CD4V1V2 district;
(4) product that step (3) is obtained finally obtains people mouse mosaic type CD4 plasmid through the three-wheel pcr amplification; First round pcr amplification primer is P3, P8, and second to take turns the pcr amplification primer be P9, P4, and third round pcr amplification primer is P3, P4.
In described step (3), the PCR condition is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of annealing 40s, 72 ℃ are extended 1min, 20 circulations; 72 ℃ were extended 8 minutes.
In described step (4), the PCR condition is: 94 ℃ of 5min; 94 ℃ of 40s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, 25 circulations; 72 ℃ were extended 8 minutes.
A kind of preparation method of people mouse mosaic type CD4 polyclonal antibody, described method comprises the steps: to use above-mentioned people mouse mosaic type CD4 plasmid DNA immunity BALB/C mice in 4 age in week, respectively the 1st, 21,35, the 49 days two hindlimb muscle injection 100 μ L 1mg/mL of mouse plasmid DNA claimed in claim 1, the wide two electrodes pin of 5mm 60V electricity irritation injection site, each electricity irritation continues 50-ms, totally 6 times, immunity zoomed in mouse serum in latter 10 days.
The people mouse mosaic type CD4 polyclonal antibody that above-mentioned method prepares.
Above-mentioned polyclonal antibody suppresses the purposes in HIV pseudovirus medicine in preparation.
A kind of carrier for expression of eukaryon, it is to utilize above-mentioned plasmid and carrier pcDNA3.1/V5-His-TOPO to build to form.
Beneficial effect of the present invention:
Process for preparing plasmid of the present invention is easy, the constructed plasmid of experiment confirm the present invention by with accessory receptor cotransfection 293T cell, can effectively mediate the HIV virus infection, inhibiting rate to the HIV pseudovirus of different subtype after 100 times of how anti-dilutions of collecting after immune mouse is 60-90%, demonstrates good wide spectrum neutralising capacity.
Exploitativeness of the present invention and outstanding substantive distinguishing features and positively effect can be embodied by following instance, but do not limit its scope.
The accompanying drawing explanation
Fig. 1 is hCD4, mhD1D2m, mhD2m and the comparison of mCD4 aminoacid sequence.Build eukaryon expression plasmid pcDNA3.1-hCD4, pcDNA3.1-mhD1D2m, pcDNA3.1-mhD2m and pcDNA3.1-mCD4 order-checking, people CD4, people mouse mosaic type CD4 and mouse CD4 aminoacid sequence are compared.
Fig. 2 mouse mosaic type plasmid DNA immunity of behaving prepares polyclonal antibody to the restraining effect (A) of HIV-1 pseudovirus with to the specific binding capacity (B) of people CD4.CD4 bonding force FACS detects the Hela negative control cell and represents with black with the front negative control sera response curve of immunity, after Hela negative control cell and the immunity of people mouse mosaic type plasmid, the sero-reaction curve represents with redness, before TZM-bl positive cell and immunity, the negative control sera response curve represents with green, and after TZM-bl positive cell and the immunity of people mouse mosaic type plasmid, the sero-reaction curve represents with blueness.
Embodiment
The structure of embodiment 1 people mouse mosaic type CD4 plasmid
1. (the Ghost cell is known cell from the Ghost cell respectively, can buy and obtain, be the clone that the HOS cytotostatic is expressed people CD4, CCR5 and CXCR4) and mouse boosting cell (cell comes from fresh mouse boosting cell) with the total RNA of TRIzol extraction; Method is as follows:
The TRIzol method is extracted cell total rna
(1) approximately 10
6Individual cell, use and add 1mL TRIzol without the RNase suction nozzle, makes to blow and beat after its effect fully;
(2) the standing 5min of room temperature, change it without in RNase 1.5mL centrifuge tube over to;
(3) add chloroform 0.2mL, piping and druming 15s, the standing 3min of room temperature;
Under (4) 4 ℃, 12000rpm is centrifugal, and 15min can see solution and be divided into three-phase.The colourless water in upper strata, be dissolved with RNA; Middle phase, be dissolved with DNA; The red phenol of lower floor/chloroform phase, be dissolved with protein;
(5) water suction is to a new 1.5mL centrifuge tube;
(6) add the 0.5mL Virahol, standing 10min under 4 ℃, join 75% ethanol 10mL simultaneously, and namely dehydrated alcohol 7.5mL adds without RNase water 2.5mL;
Under (7) 4 ℃, lower than the centrifugal 10min of 12000rpm, abandon most supernatant liquor;
(8) add 1mL75% ethanol, play even precipitation;
Under (9) 4 ℃, lower than the centrifugal 5min of 7500rpm, abandon most supernatant liquor;
(10) the 1.5mL centrifuge tube that will contain precipitation is put into 37 ℃ of incubators, the oven dry precipitation;
(11) add 13 μ L without RNase water, 57 ℃ of water-bath 10min, dissolution precipitation;
(12) add the DNase of 1 μ L without RNase, 37 ℃ of water-bath 20min, remove impurity DNA;
(13) put into 4 ℃ of refrigerators standby.
Utilize P1, P2 amplification human total rna to obtain people CD4, the total RNA of P3, P4 amplification mouse obtains mouse CD4; RT-PCR the first step reaction conditions: 42 ℃, 30min; Second step reaction conditions: 94 ℃ of 5min; 94 ℃ of sex change 50s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations; 72 ℃ are extended 10min.Finally obtain people CD4 nucleotide sequence as shown in SEQ ID No.1; Finally obtain mouse CD4 nucleotide sequence as shown in SEQ ID No.2.
2. application primer P3, P10, P11, P7, P8, P9, P4 (primer sequence is in Table 1) are through overlappingPCR amplification mouse CD4 signal peptide and the chimeric product in people CD4 V1V2 district;
2.1 take mouse CD4 as template, utilize primer P3, P10 pcr amplification mouse CD4 signal peptide, obtain sequence shown in SEQID No.3.The PCR reaction conditions: 94 ℃ 5 minutes; 94 ℃ of 30s, 55 ℃ of annealing 40s, 72 ℃ were extended 1 minute, 20 circulations; 72 ℃ were extended 8 minutes.
2.2 take people CD4 as template, utilize P11, P7PCR amplification people CD4 V1V2 district, obtain sequence shown in SEQ IDNo.4); PCR reaction system 50 μ l, in Table 1; The PCR reaction conditions: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 55 ℃ annealing 40s, 72 ℃ were extended 1 minute, 20 circulations; 72 ℃ were extended 8 minutes.
2.3 the product that utilizes above-mentioned two steps of P3, P7PCR amplification to obtain, obtain people mouse mosaic type CD4, sequence is as shown in SEQ ID No.5; PCR reaction system 50 μ l, in Table 1; The PCR reaction conditions: 94 ℃ 5 minutes; 94 ℃ 30 seconds, 55 ℃ annealing 40s, 72 ℃ were extended 1 minute, 20 circulations; 72 ℃ were extended 8 minutes.
3. through overlapping PCR, obtain people mouse mosaic type CD4 total length (the V1V2 district of people CD4 replaces the V1V2 district of mouse CD4)
3.1P3, the chimeric product of mouse CD4 signal peptide and people CD4 V1V2 district that obtains of P8 amplification step 2.3, obtain sequence as shown in SEQ ID No.6; PCR reaction system 50 μ l, in Table 1.The PCR reaction conditions: 94 ℃ 5 minutes; 94 ℃ of 40s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, 25 circulations; 72 ℃ were extended 8 minutes.
3.2 take SEQ ID No.6 as template, utilize P9, P4PCR amplification mouse CD4 V3V4 region sequence, finally obtain sequence as shown in SEQ ID No.7; PCR reaction system 50 μ l, in Table 1.The PCR reaction conditions: 94 ℃ 5 minutes; 94 ℃ of 40s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, 25 circulations; 72 ℃ were extended 8 minutes.
3.3 the product that obtains take above-mentioned two steps, as template, utilizes primer P3, P4PCR amplification to obtain people mouse mosaic type CD4 full length sequence, finally obtains people mouse mosaic type CD4 plasmid, the sequence of described plasmid is as shown in SEQ ID No.8.
Table 1PCR reaction system (50 μ L)
3.4 the people mouse mosaic type CD4 and the carrier pcDNA3.1/V5-His-TOPO that obtain are built to plasmid pcDNA3.1-mhD1D2m
By the people mouse mosaic type CD4 that obtains and carrier pcDNA3.1/V5-His-TOPO respectively through HindIII and Xho I double digestion by 1% agarose gel electrophoresis, cut glue and reclaim, the two is connected and obtains eukaryon expression plasmid pDNA3.1-mhD1D2m.
The primer sequence is in Table 2
Table 2
The preparation of embodiment 2 people mouse mosaic type CD4 polyclonal antibodies
Constructed plasmid by with auxiliary receptor CCR 5 cotransfection 293T cell, can effectively mediate the HIV virus infection.
Use Qiagen EndoFree Plasmid Maxi Kits (Qiagen, Chatsworth, CA) to extract the pcDNA3.1-mhD1D2m plasmid DNA.
Through plasmid DNA immunity BALB/C mice in 4 age in week (5/group), respectively the 1st, 21,35, the 49 days two hindlimb muscle injection 100 μ L 1mg/mL of mouse plasmid DNA of the present invention, the wide two electrodes pin of 5mm 60V electricity irritation injection site, each electricity irritation continues 50-ms, totally 6 times, before immunity, get mice serum as negative control, each immunity zoomed in mouse serum in latter 10 days and detects (HIV-1 pseudovirus and experiment) or binding ability detection (FACS) for antiviral functions.
Embodiment 3 people mouse mosaic type CD4 polyclonal antibodies suppress experiment to different subtype HIV-1 pseudovirus
In pseudovirus and experiment refer to that pseudovirus infects the reduction of luciferase reporter gene expression amount under the inhibitor effect after the Ghost cell, in order to assess the neutralising capacity of inhibitor.
The preparation of pseudovirus: by pHEP-ENV membranin plasmid and pNL4-3Luc R-E-skeleton plasmid (1g: 4g) transient transfection 293T cell.After transfection, the 293T cell is in 37 ℃ of 5% CO
2In incubator, continue to hatch 48h.Pseudovirus in the results supernatant, be stored in-80 ℃.
Pseudovirus infects and detects: 96 orifice plate every hole inoculations 1 * 10
4Individual Ghost cell, next day, cell 80% stratification, in 56 ℃ of deactivation 1h, hatched with 37 ℃, cell, pseudovirus after polyclonal antibody sample doubling dilution before serum sample experiment altogether, after 48h, detects its luciferase expression amount (RLU).In calculating and percentage or ID50.
Flow detection and analysis: 1 * 10
6TZM-b1 cell and 1: 100 dilute serum are hatched 1h on ice, the Hela cell is as negative control, after the PBS washing, with on sheep anti mouse two anti-ices of FITC mark of dilution in 1: 50, hatch 1h, cell PBS washed twice, flow cytometer showed (Becton-Dickinson, USA)
The polyvalent antibody that is obtained by the immunity of pcDNA3.1-mhD1D2m plasmid DNA has carried out the system evaluation of different subtype HIV-1 pseudovirus (strain in China) to the inhibiting rate that external HIV-1 pseudovirus infects the Ghost cell.And the polyvalent antibody be used to the every kind of pseudovirus hypotype that neutralizes has been carried out to doubling dilution, result shows that the antiserum(antisera) that obtains after the immunity of pcDNA3.1-mhD1D2m plasmid DNA all has stronger inhibiting rate, the ID of polyvalent antibody after the pcDNA3.1-mhD1D2m immunity to the multiple strain of different subtype
50540 (Fig. 2 A).
After the pcDNA3.1-mhD1D2m immunity, the specific binding of polyvalent antibody and cell surface CD4 detects further and confirms by TZM-bl and Hela cell FACS again, as shown in Fig. 2 B, preimmune serum and Hela and TZM-bl cell are without combination, after immunity polyvalent antibody (dilute 100 times) respectively with Hela and TZM-bl cell in conjunction with there were significant differences, namely many when obtaining polyvalent antibody and diluting 100 times and cell surface people CD4 still have stronger specific binding capacity.
Claims (9)
1. a people mouse mosaic type CD4 plasmid, is characterized in that, the sequence of described plasmid is as shown in SEQ ID No.8.
2. the application of the described plasmid of claim 1 in preparation mediation HIV virus infective medicament.
3. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 1, is characterized in that, described method comprises the steps:
(1) from Ghost cell and mouse boosting cell, extract total RNA respectively;
(2) utilize P1, P2 amplification human total rna to obtain people CD4, the total RNA of P3, P4 amplification mouse obtains mouse CD4;
(3) P3, P10 amplification mouse CD4 obtains mouse CD4 signal peptide; P11, P7 amplification people CD4 obtains people CD4V1V2 region sequence; Utilize P3, P7 amplification mouse CD4 signal peptide and people CD4V1V2 district to obtain mouse CD4 signal peptide and the chimeric product in people CD4V1V2 district;
(4) product that step (3) is obtained finally obtains people mouse mosaic type CD4 plasmid through the three-wheel pcr amplification; First round pcr amplification primer is P3, P8, and second to take turns the pcr amplification primer be P9, P4, and third round pcr amplification primer is P3, P4;
Described primer P1 sequence is as shown in SEQ ID No.9, primer P2 sequence is as shown in SEQ ID No.10, primer P3 sequence is as shown in SEQ ID No.11, primer P4 sequence is as shown in SEQ ID No.12, primer P7 sequence is as shown in SEQ ID No.13, and primer P8 sequence is as shown in SEQ ID No.14, and primer P9 sequence is as shown in SEQ ID No.15, primer P10 sequence is as shown in SEQ ID No.16, and primer P11 sequence is as shown in SEQ ID No.17.
4. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 3, is characterized in that, the PCR condition is in described step (3): 94 ℃ 5 minutes; 94 ℃ of 30s, 55 ℃ of annealing 40s, 72 ℃ were extended 1 minute, 20 circulations; 72 ℃ were extended 8 minutes.
5. the preparation method of people mouse mosaic type CD4 plasmid claimed in claim 3, is characterized in that, the PCR condition is in described step (4): 94 ℃ 5 minutes; 94 ℃ of 40s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, 25 circulations; 72 ℃ were extended 8 minutes.
6. a carrier for expression of eukaryon, is characterized in that, it utilizes plasmid claimed in claim 1 and carrier pcDNA3.1/V5-His-TOPO to build and forms.
7. the preparation method of a people mouse mosaic type CD4 polyclonal antibody, it is characterized in that, described method comprises the steps: that right to use requires 6 described carrier for expression of eukaryon immunity BALB/C mice in 4 age in week, respectively the 1st, 21,35, the 49 days two hindlimb muscle injection 100 μ L1mg/mL of mouse plasmid DNA claimed in claim 1, the wide two electrodes pin of 5mm 60V electricity irritation injection site, each electricity irritation continues 50-ms, and totally 6 times, immunity zoomed in mouse serum in latter 10 days.
8. the people mouse mosaic type CD4 polyclonal antibody for preparing of method claimed in claim 7.
9. polyclonal antibody claimed in claim 8 suppresses the purposes in HIV pseudovirus medicine in preparation.
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|---|---|---|---|
| CN2011102012014A CN102321653B (en) | 2011-07-18 | 2011-07-18 | Applications of man-mouse chimeric CD4 (cluster of differentiation 4) plasmid and multiclonal antibody in inhibiting HIV (human immunodeficiency virus) infection |
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| CN2011102012014A CN102321653B (en) | 2011-07-18 | 2011-07-18 | Applications of man-mouse chimeric CD4 (cluster of differentiation 4) plasmid and multiclonal antibody in inhibiting HIV (human immunodeficiency virus) infection |
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| CN102321653A CN102321653A (en) | 2012-01-18 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0512112B1 (en) * | 1990-11-27 | 1997-05-28 | Biogen, Inc. | Anti cd-4 antibodies blocking hiv-induced syncytia |
| CN101516909A (en) * | 2006-08-04 | 2009-08-26 | 巴斯德研究所 | CD4-receptor-derived peptides and method for the preparation thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0512112B1 (en) * | 1990-11-27 | 1997-05-28 | Biogen, Inc. | Anti cd-4 antibodies blocking hiv-induced syncytia |
| CN101516909A (en) * | 2006-08-04 | 2009-08-26 | 巴斯德研究所 | CD4-receptor-derived peptides and method for the preparation thereof |
Non-Patent Citations (4)
| Title |
|---|
| Nathaniel R.L..The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4.《nature》.1988,第334卷(第14期),全文. |
| The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4;Nathaniel R.L.;《nature》;19880731;第334卷(第14期);全文 * |
| 应用假病毒技术研究HIV-1复制抑制剂;曹颖莉 等;《药学学报》;20081231;第43卷(第3期);253-258 * |
| 曹颖莉 等.应用假病毒技术研究HIV-1复制抑制剂.《药学学报》.2008,第43卷(第3期),253-258. |
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