CN102618582A - Anti-p185<erbB2> human-mouse chimeric antibody lentivirus expression vector and construction method thereof - Google Patents

Abstract

The invention discloses an anti-p185<erbB2> human-mouse chimeric antibody lentivirus expression vector and a construction method thereof. Antihuman-p185<erbB2> mouse-derived monoclonal antibody variable region genes (vH and vL) and human-IgG1 constant region genes (gamma 1 and kappa) are obtained by amplification by PCR (polymerase chain reaction) process. The vH is spliced with the gamma 1 and the vL is spliced with the kappa by three-primer PCR process so as to obtain a chimeric heavy chain (H) gene and a chimeric light chain (L) gene. The chimeric heavy chain (H) gene and the chimeric light chain (L) gene are inserted onto an IRES (internal ribosome entry site) element of plasmid pVAX1/IRES downstream and upstream respectively. H-IRES-L is cut off from the pVAX1/H-IRES-L using endonucleases and is inserted into the lentivirus expression vector pWPI so as to construct a lentivirus expression pWPI/H-IRES-L. Corresponding enzyme digestion and sequencing appraisal show that the expression is consistent to that of the expected design. After transfection of cells 293T, the chimeric heavy chain (H) gene and the chimeric light chain (L) gene are co-expressed, and the chimeric antibody can be combined with p185<erbB2> molecular specificity. By the vector and the construction method, basis is laid for anti-p185<erbB2> engineering antibodies in the future.

Description

Anti-p185 ErbB2Human mouse chimeric antibody slow virus expression vector and construction process thereof

Technical field

The present invention relates to the slow virus expression vector, especially a kind of anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector and construction process thereof.

Background technology

C-erbB2/HER2/neu coded product p 185 ^ErbB2Be transmembrane glycoprotein, belong to EGF-R ELISA (EGFR) family member with tyrosine kinase activity.The amplification of c-erbB2/HER-2/neu gene and p185 ^ErbB2Proteic over-expresses is shown in multiple malignant tumour, comprises mammary cancer, ovarian cancer, colorectal carcinoma, carcinoma of endometrium, cancer of the stomach, prostate cancer and adenocarcinoma of lung.P185 ^ErbB2Over-expresses prompting tumor prognosis is poor, as shifting, recur, be prone to resistance and survival time weak point etc.Therefore, p185 ^ErbB2As tumour antigen, be an ideal oncotherapy target spot.In October, 1998, U.S. food and FAD (FDA) official approval is with the anti-p185 of a strain ^ErbB2Humanized antibody Trastuzumab (trade(brand)name Herceptin) is used for clinical treatment p185 ^ErbB2The metastatic breast cancer of high expression level.But external import antibody drug costs an arm and a leg, and how to develop the anti-p185 that has independent intellectual property right ^ErbB2Genetic engineering antibody has crucial practical significance for domestic patient, and wherein, expression vector establishment is particularly crucial.

Humanization technology to the mouse monoclonal antibody comprises the chimeric antibody of mouse monoclonal antibody constant region replacement for people's constant region at present; The part of mouse monoclonal antibody except that complementary determining region all replaced the reshaping antibody into people's sequence; The small segment antibody (comprising: Fab fragment, single-chain antibody and double-stranded antibody etc.) that antibody fragment is retrofited; Directly the antibody library of screening human immunoglobulin gene is technological from anti-storehouse.Many clinical application researchs are verified; Most of chimeric antibodies only cause extremely weak in human body even do not cause the HAMA reaction; Simultaneously chimeric antibody can keep the avidity and the specificity of parent mouse monoclonal antibody better than reshaping antibody and small molecular antibody, and the construction expression technology than reshaping antibody and phage antibody library technique more maturation, simply and cost low.

Summary of the invention

The technical problem that the present invention will solve provides a kind of anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector and construction process thereof are anti-p185 from now on ^ErbB2The fundamental research and the clinical application of chimeric antibody are laid a good foundation.

Adopt following technical scheme for solving the problems of the technologies described above: anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector, this carrier is pWPI/H-IRES-L, wherein H and L are respectively anti-p185 ^ErbB2Human mouse chimeric antibody heavy chain gene (SEQ.ID.No.2) and anti-p185 ^ErbB2Human mouse chimeric antibody light chain gene (SEQ.ID.No.1).

Above-mentioned anti-p185 ^ErbB2The construction process of human mouse chimeric antibody slow virus expression vector utilizes the PCR method to amplify anti-people p185 ^ErbB2Mouse source property monoclonal antibody variable region gene vH and vL and human IgG1's constant region gene γ 1 and κ; Adopt three-primer PCR method with vH and γ 1 again, vL and κ splice, and obtain chimeric heavy chain H and chimeric light chain L gene, the upstream and downstream of inserting the IRES element of plasmid pVAX1/IRES respectively; With restriction endonuclease H-IRES-L is downcut from pVAX1/H-IRES-L, insert among the lentiviral vectors pWPI, promptly get.

Above-mentioned anti-p185 ^ErbB2The construction process of human mouse chimeric antibody slow virus expression vector may further comprise the steps:

<1>Mouse-anti people p185 ^ErbB2Monoclonal antibody is light, heavy chain variable region gene (vL, vH) and human IgG1 gently, the amplification of weight chain constant area gene (κ, γ 1)

With plasmid pSRNC/C κ-ZC26 is template, under the effect of Taq archaeal dna polymerase, amplifies chain variable region gene vL and human IgG1's constant region of light chain gene κ of band signal peptide respectively with L-A and L-B, Kappa-A and Kappa-B; With plasmid pSRDC/C γ 1-ZC26 is template, under the effect of Taq archaeal dna polymerase, amplifies mouse monoclonal antibody heavy chain variable region gene vH and human IgG1's weight chain constant area gene γ 1 of band signal peptide respectively with H-A and H-B, gamma1-A and gamma1-B; Amplification reaction condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 ℃ are extended 1min 30s, 35 circulations, 72 ℃ are extended 10min; Reclaim test kit purifying and recovering PCR product with glue;

< 2>splicing of chimeric light chain gene L and chimeric heavy chain gene H

According to three-primer PCR method, be template with vL and κ, with the HiFiTaq archaeal dna polymerase both are spliced into chimeric light chain gene L; With vH and γ 1 is template, with the HiFiTaq archaeal dna polymerase both is spliced into chimeric heavy chain gene H; The first step: adding template vL and κ or vH and γ 1, do not add under the situation of primer, 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 1min, 10 circulations; Second step: add primer L-A and Kappa-B or H-A and gamma1-B, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min 15s, 35 circulations, 72 ℃ are extended 10min; TP-PCR splicing product chimeric light chain L or chimeric heavy chain H reclaim test kit purifying and recovering rear clone to the EZ-T carrier through glue, further sequence verification;

< 3>structure of interstitial granules pVAX1/H-IRES-L in

Cut PCR product and the plasmid PVAX1/IRES of chimeric light chain L with Nsi I and Xba I enzyme; Respectively through agarose gel electrophoresis; After the purifying and recovering, 16 ℃ of connections are spent the night under the effect of T4 ligase enzyme, connect product and transform DH-5 α competence bacterium; The extracting plasmid filters out positive colony after PCR and enzyme are cut evaluation; And then the plasmid pVAX1/IRES-L that cuts the PCR product of chimeric heavy chain H and contain chimeric light chain with BamH I and Pvu I enzyme; Make up and filter out the positive colony that contains chimeric heavy chain by above-mentioned experimental procedure; Through the gene sequencing checking, the acquisition coexpression is chimeric gently, the recombinant plasmid pVAX1/H-IRES-L of heavy chain;

< 4>structure of slow virus expression plasmid pWPI/H-IRES-L

Check order correct pVAX1/H-IRES-L plasmid with Pme I single endonuclease digestion, and the sepharose separation and purification is reclaimed test kit with gel and is reclaimed fragment H-IRES-L; Simultaneously, cut slow virus expression plasmid pWPI with the PmeI enzyme, behind the SAP dephosphorylation; Be connected with H-IRES-L with the T4DNA ligase enzyme; Connect product and transform DH-5 α competence bacterium, make PCR with carrier sequencing primer EF1 α and heavy chain downstream primer gamma1-B behind the extracting plasmid, the screening forward inserts positive colony pWPI/H-IRES-L; And carry out sequence verification, promptly get;

Above-mentioned each primer does

H-A：5’-CGC?GGA?TCC?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，

H-B：5’-GGG?GAA?GAC?CGA?TGG?GCC?CTT?GGT?GGA?TGA?GGA?GAC?GGT?GAC?CGA?GGT-3’；

gamma1-A：5’-TCC?ACC?AAG?GGC?CCA?TCG?GTC?TTC-3’，

gamma1-B：5’-ATC?GAT?CGT?CAT?TTA?CCC?GGA?GAC?AGG?GAG?AG-3’；

L-A：5’-TGC?ATG?CAT?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，

L-B：5’-GAT?GAA?GAC?AGA?TGG?TGC?AGC?CAC?AGT?GAT?CAC?CAC?CTT?GGT?CCC?CCC-3’；

Kappa-A：5’-ACT?GTG?GCT?GCA?CCA?TCT?GTC?TTC-3’，

Kappa-B：5’-GGG?TCT?AGT?CTA?ACA?CTC?TCC?CCT?GTT?GAA?GCT-3’；

EF1α：5’-TCA?AGC?CTC?AGA?CAG?TGG?TTC-3’。

The present invention has cloned anti-people p185 ^ErbB2On the basis of light, the heavy chain variable region gene of mouse monoclonal antibody, connect chimeric light, the heavy chain of people mouse, make up anti-p185 with internal ribosome entry site (IRES) ^ErbB2Human mouse chimeric antibody full-length gene, and the instrument of selecting slow virus carrier system to shift as chimeric antibody gene, thus made up anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector.Lentiviral vectors is to be the basis with the lentiviral gene group, removes it and duplicates required gene, makes up with goal gene and selected marker thing to form; Metastatic gene fragment capacity is big, nontoxicity and be difficult for bringing out host immune response, and security is better; Can not only infect somatoblast; And can the transfection terminally differentiated cells and Unseparated Cell, being integrated in the genomic goal gene of target cell can be for a long time, stably express, is usually used in gene therapy and cellular elements biological study field.The present invention makes up anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector is for the treatment research of oncogene engineered antibody is laid a good foundation.

Description of drawings

Fig. 1 is the construction strategy figure of recombinant slow virus expression vector pWPI/H-IRES-L of the present invention.

Fig. 2 is anti-people p185 ^ErbB2The PCR product electrophorogram of mouse monoclonal antibody variable region gene vL, vH and human IgG1's constant region gene κ, γ 1; Among the figure: M DNA marker DL2000; 1 contains the variable region of light chain (vL) of signal peptide; 2 contain the variable region of heavy chain (vH) of signal peptide, 3 human IgG1s' κ chain constant region, 4 human IgG1s' γ 1 chain constant region.

Fig. 3 is anti-people p185 ^ErbB2Monoclonal antibody is chimeric gently, the PCR product electrophorogram of heavy chain fusion gene, among the figure: MDNA markerDL2000,1～5 chimeric light chain gene (L), 6～10 chimeric heavy chain genes (H).

Fig. 4 is the evaluation electrophorogram of plasmid pVAX1/H-IRES-L, among the figure: M DNA marker DL10000,1pVAX1/IRES plasmid, 2pVAX1/IRES-L plasmid, 3pVAX1/H-IRES-L plasmid, the BamH I of 4pVAX1/H-IRES-L and Xba I double digestion product.

Fig. 5 is that the enzyme of slow virus expression plasmid pWPI/H-IRES-L is cut and PCR evaluation electrophorogram; Among the figure: M DNA markerDL10000,1pWPI plasmid, 2～5 and the 13pWPI/H-IRES-L plasmid; 6～7 chimeric light chain genes (L); 8～9 chimeric heavy chain genes (H), 10～11 carrier primer EF1a and heavy chain downstream primer gamma1-B are connected the pcr amplification band of plasmid to forward, and the Pme I enzyme of 12pWPI/H-IRES-L is cut product.

Fig. 6 is that the GFP of slow virus plasmid in the 293T cell expresses fluorescence microscope figure.Among the figure: the GFP of A～B empty carrier pWPI in the 293T cell expresses (the A fluorescence visual field, the B visible light visual field), and the GFP of C～D recombinant slow virus plasmid pWPI/H-IRES-L in the 293T cell expresses (the C fluorescence visual field, the D visible light visual field).

Fig. 7 is anti-p185 ^ErbB2The chimeric RT-PCR light, heavy chain expression of monoclonal antibody identifies electrophorogram, among the figure: MDNA markerDL2000,1～2 chimeric antibody light chain gene, 3～4 chimeric antibody heavy chain Fd fragments.

Embodiment

Anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector and construction process research thereof

1 materials and methods

1.1 plasmid, bacterial strain and cell

The plasmid PVAX1/IRES that contains IRES is so kind as to give by Institute of Basic Medical Sciences, A Cademy of Military Medical Sciences.Slow virus expression plasmid pWPI is that Canadian University of Ottawa clinical tumor centralab is so kind as to give.Transform and (contain anti-p185 with intestinal bacteria DH-5 α, 293T cell, plasmid pSRNC/C κ-ZC26 ^ErbB2Mouse monoclonal antibody chain variable region gene vL and human IgG1's constant region of light chain gene κ) and plasmid pSRDC/C γ 1-ZC26 (contain anti-p 185 ^ErbB2Mouse monoclonal antibody heavy chain variable region gene vH and human IgG1's weight chain constant area gene γ 1) be Guangxi Zhuang Autonomous Region treatment and prevention of tumour institute and preserve.

1.2 toolenzyme and main agents

Restriction enzyme BamH I, Pvu I, Nsi I, Xba I, Pme I, T4DNA ligase enzyme etc. are all available from Niu Yinglun biotech company.HiFiTaq archaeal dna polymerase, a small amount of plasmid extraction test kit, glue reclaim test kit, the EZ-T carrier connects test kit fast available from Genstar company.DNA marker, Pyrobest archaeal dna polymerase, shrimp alkaline phosphotase (SAP) are available from the precious biotechnology in Dalian ltd.E.Z.N.A.

no intracellular toxin plasmid extraction kit is available from OMEGA company.TRIzol reagent, LipofectamineTM2000 are available from Invitrogen company.The rt test kit is available from MBI Fermentas company.The 100mL/L foetal calf serum is available from SIJIQING biotech firm.Goat anti-human igg-HRP is available from Sigma company.People erbB2 extracellular region recombinant protein sticks up Divine Land Bioisystech Co., Ltd available from Beijing justice.The PCR primer synthesizes and the product order-checking is accomplished by Invitrogen company.

1.3 primer

Chimeric antibody is light, the amplimer of heavy chain designs according to three-primer PCR (TP-PCR) method.

The primer of the heavy chain variable region gene of amplified band signal peptide is:

H-A (SEQ.ID.No.3): 5 '-CGC GGA TCC GCC ACC ATG GGA TGG AGC TGT ATC ATC CTC T-3 ' (containing BamH I restriction enzyme site GGA TCC),

H-B (SEQ.ID.No.4): 5 '-GGG GAA GAC CGA TGG GCC CTT GGT GGA TGA GGA GAC GGT GAC CGA GGT-3 ' (wherein, human IgG1's constant region gene GGG GAA GAC CGA TGG GCC CTT GGT GGA, mouse-anti people p185 ^ErbB2Monoclonal antibody variable region gene TGA GGA GAC GGT GAC CGA GGT); The primer of amplification human IgG1 weight chain constant area gene is:

gamma1-A(SEQ.ID.No.5)：5’-TCC?ACC?AAG?GGC?CCA?TCG?GTC?TTC-3’，

Gamma1-B (SEQ.ID.No.6): 5 '-ATC GAT CGT CAT TTA CCC GGA GAC AGG GAG AG-3 ' (containing Pvu I restriction enzyme site C GAT CG);

The primer of the chain variable region gene of amplified band signal peptide is:

L-A (SEQ.ID.No.7): 5 '-TGC ATG CAT GCC ACC ATG GGA TGG AGC TGT ATC ATC CTC T-3 ' (containing Nsi I restriction enzyme site ATG CAT),

L-B (SEQ.ID.No.8): 5 '-GAT GAA GAC AGA TGG TGC AGC CAC AGT GAT CAC CAC CTT GGT CCC CCC-3 ' (wherein, human IgG1's constant region gene GAT GAA GAC AGA TGG TGC AGC CAC AGT, mouse-anti people p185 ^ErbB2Monoclonal antibody variable region gene GAT CAC CAC CTT GGT CCC CCC);

The primer of amplification human IgG1 constant region of light chain gene is:

Kappa-A(SEQ.ID.No.9)：5’-ACT?GTG?GCT?GCA?CCA?TCT?GTC?TTC-3’，

Kappa-B (SEQ.ID.No.10): 5 '-GGG TCT AGT CTA ACA CTC TCC CCT GTT GAA GCT-3 ' (containing Xba I restriction enzyme site TCT AGT);

Sequencing primer before the pWPI plasmid clone site is:

EF1 α (SEQ.ID.No.11): 5 '-TCA AGC CTC AGA CAG TGG TTC-3 '; Be used to detect anti-people p185 ^ErbB2The primer that chimeric antibody heavy chain mRNA expresses is:

H-F(SEQ.ID.No.12)：5’-AAG?TCC?AAC?TGC?AGC?AGT?CTG?G-3’，

H-R (SEQ.ID.No.13): 5 '-CTC GGG GCT GCC CTT TGG-3 '; Be used to detect anti-people p185 ^ErbB2The primer that chimeric antibody light chain mRNA expresses is:

L-F(SEQ.ID.No.14)：5’-GAC?ATT?CAG?CTG?ACC?CAG?TCT?CCA-3’，

L-R(SEQ.ID.No.15)：5’-CTA?ACA?CTC?TCC?CCT?GTT?GAA?GCT-3’。

1.4 mouse-anti people p185 ^ErbB2Monoclonal antibody is light, heavy chain variable region gene (vL, vH) and human IgG1 gently, the amplification of weight chain constant area gene (κ, γ 1)

With plasmid pSRNC/C κ-ZC26 is template, under the effect of Taq archaeal dna polymerase, amplifies chain variable region gene vL (SEQ.ID.No.16) and human IgG1's constant region of light chain gene κ (SEQ.ID.No.17) of band signal peptide respectively with L-A and L-B, Kappa-A and Kappa-B; With plasmid pSRDC/C γ 1-ZC26 is template; Under the effect of Taq archaeal dna polymerase, amplify mouse monoclonal antibody heavy chain variable region gene vH (SEQ.ID.No.18) and human IgG1's weight chain constant area gene γ 1 (SEQ.ID.No.19) of band signal peptide respectively with H-A and H-B, gamma1-A and gamma1-B, amplification reaction condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min; 56 ℃ of annealing 1min; 72 ℃ are extended 1min 30s, 35 circulations, and 72 ℃ are extended 10min; Reclaim test kit purifying and recovering PCR product with glue.

1.5 the splicing of chimeric light chain gene (L) and chimeric heavy chain gene (H)

According to three-primer PCR (TP-PCR) method, be template with vL and κ, with the HiFiTaq archaeal dna polymerase both are spliced into chimeric light chain gene (L); With vH and γ 1 is template, with the HiFiTaq archaeal dna polymerase both is spliced into chimeric heavy chain gene (H).The first step: adding template (vL and κ or vH and γ 1), do not add under the situation of primer, 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 1min, 10 circulations.Second step: add primer (L-A and Kappa-B or H-A and gamma1-B), 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min15s, 35 circulations, 72 ℃ are extended 10min.TP-PCR splicing product (chimeric light chain L or chimeric heavy chain H) reclaims test kit purifying and recovering rear clone to the EZ-T carrier through glue, further sequence verification.

1.6 the structure of middle interstitial granules pVAX1/H-IRES-L

Cut PCR product and the plasmid PVAX1/IRES of chimeric light chain L with Nsi I and Xba I enzyme; Respectively through agarose gel electrophoresis; After the purifying and recovering, 16 ℃ of connections are spent the night under the effect of T4 ligase enzyme, connect product and transform DH-5 α competence bacterium; The extracting plasmid filters out positive colony after PCR and enzyme are cut evaluation.And then the plasmid pVAX1/IRES-L that cuts the PCR product of chimeric heavy chain H and contain chimeric light chain with BamH I and Pvu I enzyme.Make up and filter out the positive colony that contains chimeric heavy chain by above-mentioned experimental procedure, through the gene sequencing checking, the acquisition coexpression is chimeric gently, the recombinant plasmid pVAX1/H-IRES-L of heavy chain.

1.7 the structure of slow virus expression plasmid pWPI/H-IRES-L

Check order correct pVAX1/H-IRES-L plasmid with Pme I single endonuclease digestion, and the sepharose separation and purification is reclaimed test kit with gel and is reclaimed fragment H-IRES-L.Cut slow virus expression plasmid pWPI with the PmeI enzyme simultaneously; Behind the SAP dephosphorylation; Be connected with H-IRES-L with the T4DNA ligase enzyme, connect product and transform DH-5 α competence bacterium, make PCR with carrier sequencing primer EF1 α and heavy chain downstream primer gamma1-B behind the extracting plasmid; The screening forward inserts positive colony pWPI/H-IRES-L, and carries out sequence verification.The construction strategy of slow virus expression plasmid pWPI/H-IRES-L is seen Fig. 1.

1.8 the expression of chimeric antibody in the 293T cell

1.8.1 the expression of green GFP GFP

According to Lipofectin ^TM2000Specification sheets is used recombinant slow virus expression vector pWPI/H-IRES-L and empty carrier pWPI transfection 293T cell respectively, is blank with the 293T cell of untransfected.48h after the transfection is through fluorescence microscope 293T cell GFP expression.

1.8.2RT-PCR detect the expression of chimeric antibody mRNA

Extracting the 293T cell total rna with the Trizol single stage method, is cDNA with total RNA rt, identifies the expression of chimeric antibody on transcriptional level with RT-PCR.With L-F is 5 ' end primer, is 3 ' end primer amplification people-mouse chimeric antibody light chain with L-R.With H-F is 5 ' end primer, is 3 ' end primer amplification people-mouse chimeric antibody heavy chain Fd fragment with H-R.

1.8.3 directly ELISA detects the expression of chimeric antibody

With 5mg/L people erbB2 extracellular region recombinant protein coated elisa plate; 4 ℃ are spent the night, and behind 37 ℃ of sealings of the confining liquid of 50g/L BSA 2h, add the metainfective 293T cell culture supernatant of pWPI/H-IRES-L and pWPI respectively; And the 293T cell conditioned medium that does not infect, be contrast with PBS simultaneously.Hatch 2h for 37 ℃; Through conventional washing, add goat anti-human igg-HRP enzyme labelled antibody and hatch, the OPD colour developing; Room temperature lucifuge reaction 5min; The enzyme linked immunological appearance is measured the A490 value, detect cell and whether secrete human mouse chimeric antibody, and whether chimeric antibody has and people erbB2 extracellular region antigen bonded specificity.Continuous data adopts mean number ± standard deviation to represent, adopts DPS 7.05 statistical softwares to carry out variance analysis.

2 results

2.1 anti-p185 ^ErbB2The mouse monoclonal antibody is light, heavy chain variable region gene and the human IgG1 is light, the amplification of weight chain constant area gene

With plasmid pSRNC/C κ-ZC26 is template, amplifies the mouse monoclonal antibody chain variable region gene vL of band signal peptide with primer L-A and L-B, the about 500bp of size; Amplify human IgG1's constant region of light chain gene κ with primer Kappa-A and Kappa-B, the about 330bp of size; With plasmid pSRDC/C γ 1-ZC26 is template, amplifies the mouse monoclonal antibody heavy chain variable region gene vH of band signal peptide with primer H-A and H-B, the about 550bp of size; Amplify human IgG1's weight chain constant area gene γ 1 with primer gamma1-A and gamma1-B, size about 1600bp (Fig. 2).

2.2 the splicing of people mouse chimeric light chain gene L and chimeric heavy chain gene H

Adopting the TP-PCR method, is template with vL and κ, is primer with L-A and Kappa-B, with the HiFiTaq enzyme both is spliced into chimeric light chain gene (L), the about 810bp of size; With vH and γ 1 is template, is primer with H-A and gamma1-B, with the HiFiTaq enzyme both is spliced into chimeric heavy chain gene (H), size about 2100bp (Fig. 3).Chimeric light, heavy chain gene after the purifying and recovering check order through Invitrogen company, and the total length sequencing result shows chimeric light, all successfully acquisitions of heavy chain gene, does not have any sudden change.

2.3 the structure of middle interstitial granules pVAX1/H-IRES-L

Chimeric light chain is cloned into plasmid pVAX1/IRES through Nsi I and Xba I double digestion; Acquisition contains the plasmid pVAX1/IRES-L of chimeric light chain; And then with chimeric heavy chain through BamH I and Pvu I double digestion human cloning plasmid pVAX1/IRES-L, obtain that coexpression is chimeric gently, the recombinant plasmid pVAX1/H-IRES-L of heavy chain.Identify that with BamH I and Xba I double digestion endonuclease bamhi is 3000bp (pVAX) and 3500bp (H-IRES-L) (see figure 4).

2.4 the structure of slow virus expression plasmid pWPI/H-IRES-L

With the correct pVAX1/H-IRES-L of order-checking with Pme I single endonuclease digestion after, cut glue and reclaim fragment H-IRES-L, and cut slow virus expression plasmid pWPI, with fragment H-IRES-L insertion cloning site with the PmeI enzyme.Correct in order to ensure H-IRES-L gene closure; Make PCR with primer EF1 α on the pWPI carrier and heavy chain downstream primer gamma1-B; If the fusion gene that inserts is a forward; PCR should amplify size and be the fragment of 2400bp, if the gene direction of insertion is reverse, then pcr amplification does not go out band.And cut evaluation with Pme I enzyme, endonuclease bamhi be 11000bp (pWPI) and 3500bp (H-IRES-L) (Fig. 5).Sequencing result shows successfully to have made up and contains anti-people p185 ^ErbB2The slow virus expression vector pWPI/H-IRES-L of human mouse chimeric antibody full-length gene.

2.5 the luciferase expression of recombinant slow virus plasmid pWPI/H-IRES-L in the 293T cell

48h after the transfection, fluorescent microscope observe the 293T cell down: the 293T cell of transfection pWPI and pWPI/H-IRES-L all has GFP to express, and untransfected 239T cell does not then have luciferase expression.Wherein, the expression amount of GFP obviously reduces (see figure 6) than the 293T cell of transfection pWPI in the 293T cell of transfection pWPI/H-IRES-L.It is thus clear that after empty carrier pWPI had inserted a bigger chimeric antibody fragment (about 3.5Kb), its transfection efficiency can reduce.

2.6 identify that from transcriptional level chimeric antibody is light, the expression of heavy chain gene

Detect through RT-PCR, behind the transfection recombined lentivirus vector pWPI/H-IRES-L, the 293T cell has chimeric light chain gene and chimeric heavy chain expression of gene (Fig. 7).

2.7 the expression of chimeric antibody and specificity are identified

Personnel selection erbB2 extracellular region antigen coated enzyme plate, the goat anti-human igg who uses the HRP mark is two anti-, carries out direct ELISA and detects.The result shows, the positive reaction of the cell conditioned medium of transfection pWPI/H-IRES-L, and the negative reaction of cell conditioned medium (seeing table 1) of the cell conditioned medium of transfection empty carrier pWPI and untransfected.This shows that secretion has chimeric antibody in the 293T cell conditioned medium behind the transfection pWPI/H-IRES-L, and chimeric antibody has and people erbB2 extracellular region antigen bonded specificity.

Table 1 is the specificity of elisa assay chimeric antibody directly

3. discuss

ErbB2 is as tumorigenic oncogene, in 20%～30% mammary cancer, crosses and expresses, and in 25%～32% ovarian cancer, crosses and expresses, and is and one of the Invasion and Metastasis of mammary cancer, ovarian cancer and the closely-related gene of prognosis.To p185 ^ErbB2The antigenic anti-p185 of extracellular region ^ErbB2Antibody has shown good antitumor curative effect.(hunman anti-murine antibodies, HMHA) reaction need be carried out genetic engineering modified to mouse source property monoclonal antibody in order to overcome the anti-rat immune globulin of the application mouse source monoclonal antibody treatment caused people of human body diseases.Multinomial research shows, makes up anti-p185 ^ErbB2Human mouse chimeric antibody is people's constant region with the replacement of mouse monoclonal antibody constant region, can intactly keep the specificity and the avidity of parent mouse monoclonal antibody, greatly reduces the heterology of mouse monoclonal antibody, suppresses high expression level p185 ^ErbB2Growth of tumour cell.

The present invention has adopted the TP-PCR method, to mouse source property p185 ^ErbB2The variable region of monoclonal antibody and human IgG1's constant region are carried out anastomosing and splicing, have obtained people mouse chimeric heavy chain and people mouse chimeric light chain, and be correct through the sequence verification splicing, do not have any sudden change.The TP-PCR method fast, conveniently, accurately.Need not to design the dna sequence dna of external source; Need not to introduce redundant restriction enzyme site base sequence; Also need not to introduce donor splicing site and splice acceptor sequence, can realize two gene Fusion splicings, produce amino acid unnecessary or variation thereby avoid introducing extra codon.Therefore, TP-PCR is variable region gene and the people's of splicing mouse the effective ways of constant region gene, in the structure human mouse chimeric antibody, has vital role.

In order to improve the expression level of chimeric antibody, the present invention has considered aspect two when making up the human mouse chimeric antibody gene.On the one hand, the constant region sequence of chimeric antibody gene is selected people's gene group sequence for use.Research shows the use genome sequence, and its expression effect is better than CDNA, and intron is an essential condition to high level expression.On the other hand, select for use IRES to make up the human mouse chimeric antibody full-length gene.The IRES element has and does not rely on cap sequence and independently raise ribosomal function, and then can start the translation of downstream gene, and two genes that are in before and after the IRES element are relatively independent, can express obtaining complete target protein.Select light, the heavy chain of IRES connection chimeric antibody for use, make the weight chain be positioned at same expression unit, avoid light, the unbalanced problem of heavy chain expression that produce owing to integration site is different.

Lentiviral vectors is to have carrier efficient, stable delivery gene ability.The pWPI that the present invention selects for use belongs to s-generation lentiviral vectors plasmid, and having removed virus replication institute in the structure must gene.Need could produce virion with packaging plasmid and coating plasmid cotransfection cell 293T, security is better.PWPI has powerful EF1-a promotor; After starting the external source destination gene expression; Under the assistance of IRES, start green fluorescence marker protein (GFP) expression of gene; Produce 2 nonfused independent albumen, can reflect the expression of goal gene indirectly, have advantages such as convenient, fast and accurate through green GFP GFP.The result shows, with the slow virus expression vector pWPI-H-IRES-L transfection 293T cell that makes up, through the expression of fluorescence microscope to green fluorescent protein, adopts RT-PCR on transcriptional level, to confirm chimeric light chain gene and chimeric heavy chain expression of gene; Directly ELISA detects and shows that chimeric antibody can combine with people erbB2 extracellular region antigen specifically.Above result shows and has successfully made up anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector is anti-p185 ^ErbB2The fundamental research and the clinical application of chimeric antibody are laid a good foundation.

Claims (3)

1. anti-p185 ^ErbB2Human mouse chimeric antibody slow virus expression vector is characterized in that this carrier is pWPI/H-IRES-L, and wherein H and L are respectively anti-p185 ^ErbB2Human mouse chimeric antibody heavy chain gene and anti-p185 ^ErbB2The human mouse chimeric antibody light chain gene.

2. according to the said anti-p185 of claim 1 ^ErbB2The construction process of human mouse chimeric antibody slow virus expression vector is characterized in that: utilize the PCR method to amplify anti-people p185 ^ErbB2Mouse source property monoclonal antibody variable region gene vH and vL and human IgG1's constant region gene γ 1 and κ; Adopt three-primer PCR method with vH and γ 1 again, vL and κ splice, and obtain chimeric heavy chain H and chimeric light chain L gene, the upstream and downstream of inserting the IRES element of plasmid pVAX1/IRES respectively; With restriction endonuclease H-IRES-L is downcut from pVAX1/H-IRES-L, insert among the lentiviral vectors pWPI, promptly get.

3. according to the said anti-p185 of claim 2 ^ErbB2The construction process of human mouse chimeric antibody slow virus expression vector is characterized in that may further comprise the steps:

<1>Mouse-anti people p185 ^ErbB2Monoclonal antibody is light, heavy chain variable region gene vL, vH and the human IgG1 is light, the amplification of weight chain constant area gene κ, γ 1

With plasmid pSRNC/C κ-ZC26 is template, under the effect of Taq archaeal dna polymerase, amplifies chain variable region gene vL and human IgG1's constant region of light chain gene κ of band signal peptide respectively with L-A and L-B, Kappa-A and Kappa-B; With plasmid pSRDC/C γ 1-ZC26 is template, under the effect of Taq archaeal dna polymerase, amplifies mouse monoclonal antibody heavy chain variable region gene vH and human IgG1's weight chain constant area gene γ 1 of band signal peptide respectively with H-A and H-B, gamma1-A and gamma1-B; Amplification reaction condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 ℃ are extended 1min 30s, 35 circulations, 72 ℃ are extended 10min; Reclaim test kit purifying and recovering PCR product with glue;

< 2>splicing of chimeric light chain gene L and chimeric heavy chain gene H

According to three-primer PCR method, be template with vL and κ, with the HiFiTaq archaeal dna polymerase both are spliced into chimeric light chain gene L; With vH and γ 1 is template, with the HiFiTaq archaeal dna polymerase both is spliced into chimeric heavy chain gene H; The first step: adding template vL and κ or vH and γ 1, do not add under the situation of primer, 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 1min, 10 circulations; Second step: add primer L-A and Kappa-B or H-A and gamma1-B, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min 15s, 35 circulations, 72 ℃ are extended 10min; TP-PCR splicing product chimeric light chain L or chimeric heavy chain H reclaim test kit purifying and recovering rear clone to the EZ-T carrier through glue, further sequence verification;

< 3>structure of interstitial granules pVAX1/H-IRES-L in

Cut PCR product and the plasmid PVAX1/IRES of chimeric light chain L with Nsi I and Xba I enzyme; Respectively through agarose gel electrophoresis; After the purifying and recovering, 16 ℃ of connections are spent the night under the effect of T4 ligase enzyme, connect product and transform DH-5 α competence bacterium; The extracting plasmid filters out positive colony after PCR and enzyme are cut evaluation; And then the plasmid pVAX1/IRES-L that cuts the PCR product of chimeric heavy chain H and contain chimeric light chain with BamH I and Pvu I enzyme; Make up and filter out the positive colony that contains chimeric heavy chain by above-mentioned experimental procedure; Through the gene sequencing checking, the acquisition coexpression is chimeric gently, the recombinant plasmid pVAX1/H-IRES-L of heavy chain;

< 4>structure of slow virus expression plasmid pWPI/H-IRES-L

Check order correct pVAX1/H-IRES-L plasmid with Pme I single endonuclease digestion, and the sepharose separation and purification is reclaimed test kit with gel and is reclaimed fragment H-IRES-L; Simultaneously, cut slow virus expression plasmid pWPI with the PmeI enzyme, behind the SAP dephosphorylation; Be connected with H-IRES-L with the T4DNA ligase enzyme; Connect product and transform DH-5 α competence bacterium, make PCR with carrier sequencing primer EF1 α and heavy chain downstream primer gamma1-B behind the extracting plasmid, the screening forward inserts positive colony pWPI/H-IRES-L; And carry out sequence verification, promptly get;

Said each primer does

H-A：5’-CGC?GGA?TCC?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，

H-B：5’-GGG?GAA?GAC?CGA?TGG?GCC?CTT?GGT?GGA?TGA?GGA?GAC?GGT?GAC?CGA?GGT-3’；

gamma1-A：5’-TCC?ACC?AAG?GGC?CCA?TCG?GTC?TTC-3’，

gamma1-B：5’-ATC?GAT?CGT?CAT?TTA?CCC?GGA?GAC?AGG?GAG?AG-3’；

L-A：5’-TGC?ATG?CAT?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，

L-B：5’-GAT?GAA?GAC?AGA?TGG?TGC?AGC?CAC?AGT?GAT?CAC?CAC?CTT?GGT?CCC?CCC-3’；

Kappa-A：5’-ACT?GTG?GCT?GCA?CCA?TCT?GTC?TTC-3’，

Kappa-B：5’-GGG?TCT?AGT?CTA?ACA?CTC?TCC?CCT?GTT?GAA?GCT-3’；

EF1α：5’-TCA?AGC?CTC?AGA?CAG?TGG?TTC-3’。

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