CN102618583A - Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector - Google Patents
Lentivirus expression vector of anti-p185erdB2 human mouse chimeric antibody containing F/2A sequence and construction method of lentivirus expression vector Download PDFInfo
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Abstract
The invention discloses a lentivirus expression vector of an anti-p185erdB2 human mouse chimeric antibody containing an F/2A sequence, and a construction method of the lentivirus expression vector. Furin or foot and mouth disease virus/2A polypeptide (F/2A) with self shearing capacity is connected with a heavy chain and a light chain of the human-mouse chimeric antibody to form an open reading frame (ORF), and the ORF is inserted into a lentivirus expression vector pWPI to construct the expression vector pWPI/H-F2A-L of the recombination anti-p185erdB2 overall-length human mouse chimeric antibody. According to sequencing identification, the pWPI/H-F2A-L is consistent to expected design. The infection efficiency of recombination lentivirus is 87.68%, and the expressions of the chimeric heaving chain and light chain are formed after the recombination lentivirus is infected with 293T cells. Compared with that of a lentivirus expression vector of an IRES (Internal Ribosome Entry Site) chimeric antibody, the expression level of the chimeric antibody mediated by the F/2A is higher than that of the chimeric antibody mediated by IRES, thereby laying a foundation for research on anti-p185erdB2 engineering antibodies.
Description
Technical field
The present invention relates to the slow virus expression vector, especially a kind of anti-p185 that contains the F/2A sequence
ErbB2Human mouse chimeric antibody slow virus expression vector and construction process thereof.
Background technology
C-erbB2/HER2/neu coded product p185
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.P185
ErbB2As tumour antigen, be an ideal oncotherapy target spot.U.S. food and FAD (FDA) in official approval in October, 1998 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.
The main mode of carrier construction expressed antibody molecule has three types at present: the one, light, heavy chain gene are connected to different expression vectors respectively; The 2nd, light, heavy chain gene is on identical carrier but be in different expression unit; The 3rd, connect light, heavy chain with internal ribosome entry site (IRES), make the weight chain be in same expression unit.But there are some defectives in these modes, and are low like the initial efficient of being expressed of gene; Phase mutual interference between the promotor; The expression imbalances of upstream gene and downstream gene etc. cause light, heavy chain expression level to have significant difference, cause complete antibody developed by molecule level lower.
Summary of the invention
The technical problem that the present invention will solve provides a kind of anti-p185 of the F/2A of containing sequence
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.
The anti-p185 that contains the F/2A sequence
ErbB2Human mouse chimeric antibody slow virus expression vector, this carrier is pWPI/H-F2A-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), F2A are that furin and foot and mouth disease virus 2A shear polypeptide (SEQ.ID.No.15) certainly.
Anti-p185
ErbB2The human mouse chimeric antibody heavy chain gene is by mouse-anti people p185
ErbB2Monoclonal antibody heavy chain variable region gene vH (SEQ.ID.No.13) and human IgG1's weight chain constant area gene γ 1 (SEQ.ID.No.14) is formed by connecting; Anti-p185
ErbB2The human mouse chimeric antibody light chain gene is by mouse-anti people p185
ErbB2Monoclonal antibody chain variable region gene vL (SEQ.ID.No.16) and human IgG1's constant region of light chain gene κ (SEQ.ID.No.17) is formed by connecting.
The above-mentioned anti-p185 that contains the F/2A sequence
ErbB2The construction process of human mouse chimeric antibody slow virus expression vector: the heavy chain and the light chain that connect human mouse chimeric antibody with furin Furin/ foot and mouth disease virus 2A polypeptide F/2A with self-shear ability; Form an ORFs ORF; Insert slow virus expression vector pWPI, promptly get.
The above-mentioned anti-p185 that contains the F/2A sequence
ErbB2The construction process of human mouse chimeric antibody slow virus expression vector may further comprise the steps:
<1>Anti-p185
ErbB2Human mouse chimeric antibody is heavy, the amplification of light chain gene
Contain anti-p185 with the extracting of a small amount of plasmid extraction test kit
ErbB2Human mouse chimeric antibody is light, the plasmid pWPI/H-IRES-L of heavy chain gene, and as template, under the effect of DNA Taq enzyme, amplifies chimeric heavy chain gene H and chimeric light chain gene L with H-A and gamma1-B, L-A and Kappa-B respectively; Amplification condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations, last 72 ℃ are extended 10min; The PCR product reclaims test kit with glue and reclaims goal gene behind 1% agarose gel electrophoresis;
< 2>structure of total length chimeric antibody H-F2A-L
1. synthetic this fragment of linker fragment F2A is made up of 3 parts: 3 ' terminal sequence, furin and the foot and mouth disease virus 2A that contains the chimeric heavy chain H of NsiI restriction enzyme site is from shearing peptide sequence and containing the 5 ' terminal sequence of the chimeric light chain L of PvuII restriction enzyme site;
2. the structure of middle interstitial granules pUC57/F2A is inserted into linker fragment F2A between the BamH I and Xba I of pUC57 plasmid, interstitial granules pUC57/F2A in the acquisition;
3. the structure of plasmid pUC57/F2A-L that contains chimeric light chain is with BamH I and Xba I double digestion plasmid pUC57/F2A, and enzyme is cut product behind 1% agarose gel electrophoresis, reclaims test kit with glue and reclaims linker fragment F2A and carrier segments pUC57 respectively; And then with a Pvu II enzyme cut-grafting fragment F2A, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the linker fragment F2A of Pvu II restriction enzyme site; With Pvu II and Xba I double digestion chimeric light chain L, obtaining 5 ' end is that Pvu II restriction enzyme site and 3 ' end are the light chain segments L in Xba I site; Then, these two fragments are connected rear clone with the T4DNA ligase enzyme go into carrier pUC57, obtain to contain the plasmid pUC57/F2A-L of chimeric light chain;
4. the splicing of fragment H-F2A-L is cut pUC57/F2A-L with BamH I enzyme and Xba I enzyme, obtains fragment F2A-L; And then with Nsi I endonuclease bamhi F2A-L, obtaining 5 ' end is that Nsi I restriction enzyme site and 3 ' end are the fragment F2A-L of Xba I restriction enzyme site; With BamH I and Nsi I double digestion chimeric heavy chain H, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the heavy chain fragment H in Nsi I site; Link to each other with fragment F2A-L through the T4DNA ligase enzyme, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the chimeric antibody full length fragment H-F2A-L (SEQ.ID.No.3) in Xba I site;
< 3>structure of slow virus expression plasmid pwpI/H-F2A-L
With the fragment H-F2A-L of purifying and recovering, mend flat two ends with DNA end-filling test kit; Cut slow virus expression plasmid pWPI with the PmeI enzyme simultaneously; Behind the SAP dephosphorylation, be connected with H-F2A-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-F2A-L, and carries out sequence verification, promptly gets;
Above-mentioned each primer does
H-A(SEQ.ID.No.4)：5’-CGC?GGA?TCC?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，
gamma1-B(SEQ.ID.No.5)：5’-ATC?GAT?CGT?CAT?TTA?CCC?GGA?GAC?AGG?GAG?AG-3’：
L-A(SEQ.ID.No.6)：5’-TGC?ATG?CAT?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-3’，
Kappa-B(SEQ.ID.No.7)：5’-GGG?TCT?AGT?CTA?ACA?CTC?TCC?CCT?GTT?GAA?GCT-3’：
EF1α(SEQ.ID.No.8)：5’-TCA?AGC?CTC?AGA?CAG?TGG?TTC-3’。
(Foot-and-mouth disease virus, polypeptide 2A genes encoding FMDV) is a kind of from shear protein for foot and mouth disease virus.Be connected to form a complete ORF with the 2A sequence between two foreign genes; Fusion rotein can be cut by 2A at the C end in coding 2A zone during translation; With the fusion rotein cutting and separating is two independently albumen, and short gene order of FMDV 2A and 2A make it to become the effective tool that makes up the polycistron carrier from shear efficiency efficiently.People such as Jianmin F. utilize furin (the Furin)/foot and mouth disease virus 2A polypeptide (F/2A) of self-shear ability to express heavy chain and the light chain of monoclonal antibody DC101 simultaneously in glandular associated virus expression vector; Thereby realized full length antibody sustainable high efficiency expression in vivo, and obtained significant antitumous effect.The present invention is light at chimeric antibody, introduce the F/2A sequence between the heavy chain and the slow virus expression system carries out coexpression to light, heavy chain; And compare with the chimeric antibody slow virus expression vector that contains IRES, the result shows that the expression level by the chimeric antibody of F/2A mediation will be higher than the chimeric antibody by the IRES mediation.The present invention has showed new construction strategy for the complete antibody that obtains high expression level, also is anti-p185
ErbB2New approach has been opened up in the research of genetic engineering antibody.
Description of drawings
Fig. 1 is the construction strategy figure of recombinant slow virus expression vector pWPI/H-F2A-L of the present invention.
Fig. 2 is anti-people p185
ErbB2Monoclonal antibody is chimeric gently, the PCR product electrophorogram of heavy chain gene, among the figure: M DNA marker DL2000,1～5 chimeric light chain gene (L), 6～10 chimeric heavy chain genes (H).
Fig. 3 is a fragment H-F2A-L electrophorogram, among the figure: M DNA marker DL10000,1 fragment H-F2A-L.
Fig. 4 is that the PCR of slow virus expression plasmid pWPI/H-F2A-L identifies electrophorogram; Among the figure: M DNA marker DL10000; 1～3pWPI/H-F2A-L plasmid; The 4pWPI plasmid, 5 are connected the pcr amplification band of plasmid with 7 carrier primer EF1a and heavy chain downstream primer gamma1-B to forward, 6 with 8 carrier primer EF1a and heavy chain downstream primer gamma1-B to the reverse pcr amplification band that is connected plasmid.
Fig. 5 is the microscopic examination figure of slow virus three plasmid co-transfection 293T cell egfp expressions; Among the figure: A, C, E are respectively pWPI/H-F2A-L group under the fluorescence, pWPI/H-IRES-L group, pWPI group, and B, D, F are respectively pWPI/H-F2A-L group under the visible light, pWPI/H-IRES-L group, pWPI group.
Fig. 6 is the transfection efficiency flow cytometer detection figure of 3 kinds of slow viruss, and among the figure: A, B, C are respectively the 293T cells of transfection pWPI/H-F2A-L, pWPI/H-IRES-L, pWPI, and D is the 293T cell of untransfected.
Fig. 7 is the expression electrophorogram that RT-PCR identifies light, the heavy chain of chimeric antibody; Among the figure: the light chain gene of 1 chimeric antibody H-F2A-L; The light chain gene of 2 chimeric antibody H-IRES-L, the heavy chain Fd fragment of 3 chimeric antibody H-F2A-L, the heavy chain Fd fragment of 4 chimeric antibody H-IRES-L.
Embodiment
The anti-p185 that contains the F/2A sequence
ErbB2Human mouse chimeric antibody slow virus expression vector and construction process research thereof
1 materials and methods
1.1 plasmid, bacterial strain and cell
Three plasmid slow virus systems are made up of expression plasmid pWPI, structure plasmid pCMV-dR 8.74 and big envelope plasmid pMD2G, for Canadian University of Ottawa clinical tumor centralab is so kind as to give.The plasmid pUC57/F2A that contains the F/2A sequence gives birth to worker company by Shanghai and makes up.Transform with intestinal bacteria DH-5 α, contain anti-p185
ErbB2The plasmid pWPI/H-IRES-L of chimeric light, the heavy chain gene of people mouse, 293T cell are by be the preservation of Guangxi Zhuang Autonomous Region treatment and prevention of tumour institute.
1.2 toolenzyme and main agents
Restriction enzyme BamH I, Pvu II, Nsi I, Xba I, Pme I, T4DNA ligase enzyme etc. are all available from Niu Yinglun biotech company.Taq archaeal dna polymerase, a small amount of plasmid extraction test kit, glue reclaim test kit, DNA end-filling test kit all available from Genstar company.DNA marker, 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; RIzol reagent is available from Invitrogen company.Goat-anti people κ antibody, goat anti-human igg Fc-HRP, the anti-sheep IgG-HRP of rabbit are available from Sigma company.The rt test kit is available from MBI Fermentas company.The 100mL/L foetal calf serum is available from SIJIQING biotech firm.The PCR primer is synthetic and the product order-checking is synthetic by Invitrogen company.
1.3 primer
The primer of amplification chimeric antibody heavy chain gene is:
H-A (SEQ.ID.No.4): 5 '-CGC GGA TCC GCC ACC ATG GGA TGG AGC TGT ATC ATC CTCT-3 ' (containing BamH I restriction enzyme site GGA TCC),
gamma1-B(SEQ.ID.No.5)：5’-ATC?GAT?CGT?CAT?TTA?CCC?GGA?GAC?AGG?GAG?AG-3’；
The primer of amplification chimeric antibody light chain gene is:
L-A(SEQ.ID.No.6)：5’-TGC?ATG?CAT?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTCT-3’，
Kappa-B(SEQ.ID.No.7)：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:
EF1α(SEQ.ID.No.8)：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.9)：5’-AAG?TCC?AAC?TGC?AGC?AGT?CTG?G-3’
H-R(SEQ.ID.No.10)：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.11)：5’-GAC?ATT?CAG?CTG?ACC?CAG?TCT?CCA-3’，
L-R(SEQ.ID.No.12)：5’-CTA?ACA?CTC?TCC?CCT?GTT?GAA?GCT-3’。
1.4 anti-p185
ErbB2Human mouse chimeric antibody is heavy, the amplification of light chain gene
Contain anti-p185 with the extracting of a small amount of plasmid extraction test kit
ErbB2Human mouse chimeric antibody is light, the plasmid pWPI/H-IRES-L of heavy chain gene, and as template, under the effect of DNA Taq enzyme, amplifies chimeric heavy chain gene H and chimeric light chain gene L with H-A and gamma1-B, L-A and Kappa-B respectively; Amplification condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations, last 72 ℃ are extended 10min; The PCR product reclaims test kit with glue and reclaims goal gene behind 1% agarose gel electrophoresis.
1.5 the structure of total length chimeric antibody H-F2A-L
1.5.1 linker fragment F2A's is synthetic
Give birth to the worker synthetic linker fragment F2A of company by Shanghai, this fragment is made up of 3 parts: the 3 ' terminal sequence of chimeric heavy chain H (containing Nsi I restriction enzyme site), furin and foot and mouth disease virus 2A are from the 5 ' terminal sequence of shearing peptide sequence and chimeric light chain L (containing the PvuII restriction enzyme site);
1.5.2 the structure of middle interstitial granules pUC57/F2A
Linker fragment F2A is inserted between the BamH I and Xba I of pUC57 plasmid interstitial granules pUC57/F2A in the structure;
1.5.3 contain the structure of the plasmid pUC57/F2A-L of chimeric light chain
With BamH I and Xba I double digestion plasmid pUC57/F2A, enzyme is cut product behind 1% agarose gel electrophoresis, reclaims test kit with glue and reclaims linker fragment F2A and carrier segments pUC57 respectively; And then with a Pvu II enzyme cut-grafting fragment F2A, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the linker fragment F2A of Pvu II restriction enzyme site; With Pvu II and Xba I double digestion chimeric light chain L, obtaining 5 ' end is that Pvu II restriction enzyme site and 3 ' end are the light chain segments L in Xba I site; Then, these two fragments are connected rear clone with the T4DNA ligase enzyme go into carrier pUC57, obtain to contain the plasmid pUC57/F2A-L of chimeric light chain;
1.5.4 the splicing of fragment H-F2A-L
Cut pUC57/F2A-L with BamH I enzyme and Xba I enzyme, obtain fragment F2A-L; And then with Nsi I endonuclease bamhi F2A-L, obtaining 5 ' end is that Nsi I restriction enzyme site and 3 ' end are the fragment F2A-L of Xba I restriction enzyme site; With BamH I and Nsi I double digestion chimeric heavy chain H, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the heavy chain fragment H in Nsi I site; Link to each other with fragment F2A-L through the T4DNA ligase enzyme, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the chimeric antibody full length fragment H-F2A-L in Xba I site;
1.6 the structure of slow virus expression plasmid pWPI/H-F2A-L
With the fragment H-F2A-L of purifying and recovering, mend flat two ends with DNA end-filling test kit.Cut slow virus expression plasmid pWPI with the PmeI enzyme simultaneously; Behind the SAP dephosphorylation; Be connected with H-F2A-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-F2A-L, and carries out sequence verification.The construction strategy of slow virus expression plasmid pWPI/H-F2A-L is seen Fig. 1.
1.7 packing of slow virus and virus titer are measured
With the calcium phosphate precipitation method with slow virus pWPI/H-F2A-L carrier, pCMV-dR8.74, pMD2.G cotransfection 293T cell.Under fluorescent microscope, observe the luciferase expression situation behind the 48h.72h collects the viral supernatant after the transfection, filters with 0.45 μ m filter, and the nutrient solution supernatant of acquisition then contains the recombinant virus that carries H-F2A-L.Transfection pWPI/H-IRES-L+pCMVdR8.74+pMD2.G and pWPI+pCMVdR8.74+pMD2.G (unloaded vehicle Control) entering 293T cell is made comparisons respectively simultaneously.
In 6 orifice plates, inoculate 2 * 10 respectively
5The 293T cell, with collected supernatant by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Doubly dilution joins the supernatant after the 1ml dilution respectively in the corresponding hole.Under fluorescent microscope, observe expression and the counting of GFP in each concentration hole behind the 48h and express the number of cells of GFP, multiply by corresponding extension rate and can obtain virus titer.
1.8 infecting the transduction efficiency of 293T cell, recombinant slow virus detects
Press method (Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood [J] the .Mol Ther of Hanawa H with recombinant slow virus; 2002; 5:242-251) infect the 293T cell respectively; Be divided into experiment one group of (pWPI/H-F2A-L), experiment two groups of (pWPI/H-IRES-L) and control groups (pWPI); Behind the recombinant virus infection 48h, detect GFP positive cell percentage ratio with flow cytometer: with 1 * 10
6The cell centrifugation deposition; Wash secondary with PBS cell is suspended among the 1ml PBS again, blow and beat into individual cells as far as possible, statistics GFP positive cell number on flow cytometer; Detect slow virus infection efficient (transgene efficiency), not infect the negative contrast of 293T cell of recombinant slow virus.
1.9 the expression of chimeric antibody in the 293T cell
1.9.1RT-PCR detect the expression of chimeric antibody
Infect 3 groups of 293T cells respectively with 3 kinds of slow viruss once more, the 293T cell after the collection superinfection extracts total RNA, and reverse transcription becomes cDNA, identifies the stably express of chimeric antibody H-F2A-L and H-IRES-L respectively with RT-PCR.With H-F is 5 ' end primer, is 3 ' end primer amplification people-mouse chimeric antibody heavy chain Fd fragment with H-R; With L-F is 5 ' end primer, is 3 ' end primer amplification people-mouse chimeric antibody light chain with L-R.
1.9.2ELISA method detects the expression of chimeric antibody
With 1mg/L goat-anti people κ chain antibody coated elisa plate; 4 ℃ are spent the night; Behind 37 ℃ of sealings of 50g/L BSA confining liquid 2h; Add the metainfective 293T cell culture supernatant of recombinant slow virus (pWPI/H-F2A-L, pWPI/H-IRES-L and pWPI) respectively, simultaneously the negative contrast of 293T cell conditioned medium not infect.Hatch 2h for 37 ℃,, add goat anti-human igg Fc fragment-HRP enzyme labelled antibody and hatch through conventional washing, the OPD colour developing, room temperature lucifuge reaction 5min, the enzyme linked immunological appearance is measured the A490 value, detects cell and whether secretes people-mouse chimeric antibody.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-people p185
ErbB2The amplification of monoclonal antibody chimeric heavy chain gene (H) and chimeric light chain gene (L)
With primer H-A and gamma1-B, L-A and Kappa-B, from plasmid pWPI/H-IRES-L, amplify p185 respectively
ErbB2The chimeric heavy chain gene (H) of monoclonal antibody and chimeric light chain gene (L).Identify that through 1% agarose gel electrophoresis these two specific bands of H and L are consistent with theoretical value, size is respectively (like Fig. 2) about 2100bp and 810bp.
2.2 the structure of total length chimeric antibody H-F2A-L
Utilize middle interstitial granules pUC57/F2A that makes up and chimeric heavy chain gene H, the chimeric light chain gene L that amplifies; Cut, connect through the multistep enzyme; The final chimeric antibody full length fragment H-F2A-L that obtains, the visible band (Fig. 3) that is approximately 3000bp behind purifying and recovering, the electrophoresis.
2.3 the structure of slow virus expression plasmid pWPI/H-F2A-L
Cut slow virus expression plasmid pWPI with the PmeI enzyme, with inserting cloning site behind the fragment H-F2A-L end-filling.Correct in order to ensure H-F2A-L gene closure; Make PCR with primer EF1 α on the pWPI carrier and heavy chain downstream primer gamma1-B; If the 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 (Fig. 4).Sequencing result shows and has successfully made up the anti-p185 that contains the F/2A sequence
ErbB2The slow virus expression vector pWPI/H-F2A-L of human mouse chimeric antibody full-length gene.
2.4 packing of slow virus and virus titer are measured
With slow virus system three plasmid co-transfection 293T packing cells, under fluorescent microscope, to observe behind the 48h, three experimental group are all it is thus clear that the expression (Fig. 5) of a large amount of green fluorescent protein GFP explains that the package cell line that makes up can produce slow virus effectively.Packing cell supernatant behind the transfection 72h is infected 293T cell (this moment, 293T was as target cell) again, measure the titre of slow virus in 6 orifice plates behind the 48h, the virus titer that the result contrasts empty carrier pWPI group is the highest, is 2.1 * 10
6TU/mL, the virus titer of pWPI/H-F2A-L group takes second place, and is 4.3 * 10
5TU/mL; And the virus titer of pWPI/H-IRES-L group is minimum, is 3.5 * 10
5TU/mL.
2.5 the detected result of gene transfer efficient
Infect the 293T cell respectively with 3 kinds of recombinant slow virus, detect GFP positive cell percentage ratio through flow cytometer behind the 48h.The result shows; The positive cell rate of pWPI/H-F2A-L group is 87.68%, and the positive cell rate of pWPI/H-IRES-L group is 79.08%, and the positive cell rate of empty carrier pWPI group is the highest; Be 95.51%, the 293T negative cells that does not infect slow virus does not have luciferase expression (Fig. 6).It is thus clear that after inserting chimeric antibody gene among the empty carrier pWPI, the transfection efficiency of slow virus can descend, and the efficient of transduction chimeric antibody H-F2A-L gene will be higher than the efficient of transduction chimeric antibody H-IRES-L gene.
2.6 chimeric antibody is light, the expression of heavy chain gene mRNA
Detect through RT-PCR, chimeric light chain gene and chimeric heavy chain expression of gene (Fig. 7) are all arranged behind the 293T cell of transfection pWPI/H-F2A-L and the 293T cell of transfection pWPI/H-IRES-L.
2.7 the expression of chimeric antibody
Adopt the sandwich ELISA method to measure the expression of chimeric antibody in 4 groups of cells (the 293T cell of the 293T cell of transfection pWPI/H-F2A-L, the 293T cell of transfection pWPI/H-IRES-L, transfection pWPI and the 293T cell of untransfected) supernatant.The result shows, the positive reaction of cell conditioned medium of the cell conditioned medium of transfection pWPI/H-F2A-L and transfection pWPI/H-IRES-L, and the negative reaction of cell conditioned medium (table 1) of transfection empty carrier pWPI.This shows no matter be that weight, the light chain that F/2A element or IRES element connect can both be expressed, and can both correctly be assembled into activated antibody in the 293T cell, be secreted into the extracellular well.Wherein, the expression level by the chimeric antibody of F/2A mediation will be higher than the chimeric antibody by the IRES mediation.
The expression of table 1 elisa assay chimeric antibody
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.In order to overcome the problem that mouse monoclonal antibody occurs in human body therapy, classical genetic engineering antibody technology promptly makes up human mouse chimeric antibody with the constant region of the alternative murine antibody of human antibody constant region.Chimeric antibody has not only kept the specificity and the avidity of parental antibody; Greatly reduce HAMA (HAMA) reaction; And its people source Fc section can excite antibody-mediated cell toxicant (ADCC) to act on and cell toxicant (CDC) effect of complement-mediated, thereby increase its fragmentation effect to tumour cell.
Make up the strategy of chimeric antibody expression cassette, existing will be light, heavy chain is implemented in different expression vectors respectively, also have light, heavy chain are series at same expression vector.It is reported that the bicistronic mRNA carrier not only can carry out coupling with expression light, heavy chain, but also can improve the expression level of antibody.The element of expression bicistronic mRNA commonly used is internal ribosome entry site (IRES) or shears polypeptide 2A certainly; 1. their mechanism of action be respectively 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; 2. the 2A element higher structure that then can in translation process, form causes sterically hindered to the ribosomal peptidyl transferase center; Causing forming normal peptide chain connects; But simultaneously rrna can continue to translate downstream albumen, thereby the effect that forms a kind of albuminoid lytic enzyme is with former and later two albumen cis " incision ".Because the difference of both mechanisms of action; Also there are tangible characteristics in the actual effect that causes them in polycistronic expression, to bring into play; Therefore in practical application, need to make up two or more different expression vectors usually and compare, just can find only expressional scheme.
2005, people such as Jianmin F sheared light chain and the heavy chain that polypeptide (F/2A) is connected antibody through utilizing furin (Furin) restriction enzyme site with 2A certainly, make up glandular associated virus expression vector.Furin is a kind of important endo-protease in the eukaryotic cells.Furin can discern specific aminoacid sequence, and many important polypeptide and proteic precursor in the Secretory Pathway are sheared processing, makes it biologically active.Cut from the enzyme of shearing and Furin restriction enzyme site through the 2A fragment; Light, the heavy chain of a full length antibody have been realized in an expression vector and ORFs, balancedly expressing simultaneously; Automatic shearing through F/2A; Removed and connected all exogenous arrays light, heavy chain; The antibody expression level has reached the level of treatment tumour, has overcome light, heavy chain and has been structured in the antibody expression that is caused in two carriers and matees incomplete shortcoming, for the expression of full length antibody provides a new method and technology.2011; Li Meng etc. utilize F/2A to make up reorganization anti-death receptor 5 (DR5) total length human mouse chimeric antibody slow virus expression vector pWPXL-HF2AL; The result shows with the human mouse chimeric antibody of F/2A polypeptide technological expression and can shear the oneself of F/2A place, human mouse chimeric antibody light, heavy chain expression is symmetrical and have the avidity similar with its maternal mouse source antibody and kill and wound the activity of kinds of tumor cells.
On this basis, the present invention utilizes F/2A to make up the anti-p185 of reorganization
ErbB2Total length human mouse chimeric antibody slow virus expression vector pWPI/H-F2A-L, and compare with another two-cistron expression vector pWPI/H-IRES-L that has made up.The result shows, the virus titer (4.3 * 10 of pWPI/H-F2A-L group
5TU/mL) be higher than the virus titer (3.5 * 10 that pWPI/H-IRES-L organizes
5TU/mL), and the efficiency of infection of preceding papova (87.68%) also be higher than (79.08%) of back group.Its reason possibly be because the structure of IRES big (about 590bp), and the structure of F/2A little (84bp) so F/2A can be alleviated the pressure of lentiviral vectors pWPI tonburden, more helps viral packing and infection; ELISA detects demonstration, will be higher than by the IRES mediation by the chimeric antibody expression level of F2A mediation.Its reason possibly be because IRES to be positioned at thereafter the initial ability of gene translation relatively a little less than; The expression amount that causes its downstream gene only is 20%～50% of the upper reaches; And F/2A is superior to the IRES element aspect the upstream and downstream genetic expression balance keeping, so can be higher by the complete developed by molecule level of chimeric antibody of F/2A mediation.In sum, contain the chimeric antibody expression cassette of F/2A, compare, more help the transgenosis of slow virus mediation and the expression of chimeric antibody with the expression cassette that contains IRES.
Claims (4)
1. anti-p185 who contains the F/2A sequence
ErbB2Human mouse chimeric antibody slow virus expression vector is characterized in that this carrier is pWPI/H-F2A-L, and wherein H and L are respectively anti-p185
ErbB2Human mouse chimeric antibody heavy chain gene and anti-p185
ErbB2Human mouse chimeric antibody light chain gene, F2A are that furin and foot and mouth disease virus 2A shear polypeptide certainly.
2. the anti-p185 that contains the F/2A sequence according to claim 1
ErbB2Human mouse chimeric antibody slow virus expression vector is characterized in that: said anti-p185
ErbB2The human mouse chimeric antibody heavy chain gene is by mouse-anti people p185
ErbB2Monoclonal antibody heavy chain variable region gene vH and human IgG1's weight chain constant area gene γ 1 is formed by connecting; Said anti-p185
ErbB2The human mouse chimeric antibody light chain gene is by mouse-anti people p185
ErbB2Monoclonal antibody chain variable region gene vL and human IgG1's constant region of light chain gene κ is formed by connecting.
3. according to the said anti-p185 that contains the F/2A sequence of claim 1
ErbB2The construction process of human mouse chimeric antibody slow virus expression vector; It is characterized in that: the heavy chain and the light chain that connect human mouse chimeric antibody with furin Furin/ foot and mouth disease virus 2A polypeptide F/2A with self-shear ability; Form an ORFs ORF; Insert slow virus expression vector pWPI, promptly get.
4. according to the said anti-p185 that contains the F/2A sequence of claim 3
ErbB2The construction process of human mouse chimeric antibody slow virus expression vector is characterized in that may further comprise the steps:
<1>Anti-p185
ErbB2Human mouse chimeric antibody is heavy, the amplification of light chain gene
Contain anti-p185 with the extracting of a small amount of plasmid extraction test kit
ErbB2Human mouse chimeric antibody is light, the plasmid pWPI/H-IRES-L of heavy chain gene, and as template, under the effect of DNA Taq enzyme, amplifies chimeric heavy chain gene H and chimeric light chain gene L with H-A and gamma1-B, L-A and Kappa-B respectively; Amplification condition is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min, and 56 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations, last 72 ℃ are extended 10min; The PGR product reclaims test kit with glue and reclaims goal gene behind 1% agarose gel electrophoresis;
< 2>structure of total length chimeric antibody H-F2A-L
1. synthetic this fragment of linker fragment F2A is made up of 3 parts: 3 ' terminal sequence, furin and the foot and mouth disease virus 2A that contains the chimeric heavy chain H of Nsi I restriction enzyme site is from shearing peptide sequence and containing the 5 ' terminal sequence of the chimeric light chain L of Pvu II restriction enzyme site;
2. the structure of middle interstitial granules pUC57/F2A is inserted into linker fragment F2A between the BamH I and Xba I of pUC57 plasmid, interstitial granules pUC57/F2A in the acquisition;
3. the structure of plasmid pUC57/F2A-L that contains chimeric light chain is with BamH I and Xba I double digestion plasmid pUC57/F2A, and enzyme is cut product behind 1% agarose gel electrophoresis, reclaims test kit with glue and reclaims linker fragment F2A and carrier segments pUC57 respectively; And then with a Pvu II enzyme cut-grafting fragment F2A, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the linker fragment F2A of Pvu II restriction enzyme site; With Pvu II and Xba I double digestion chimeric light chain L, obtaining 5 ' end is that Pvu II restriction enzyme site and 3 ' end are the light chain segments L in Xba I site; Then, these two fragments are connected rear clone with the T4DNA ligase enzyme go into carrier pUC57, obtain to contain the plasmid pUC57/F2A-L of chimeric light chain;
4. the splicing of fragment H-F2A-L is cut pUC57/F2A-L with BamH I enzyme and Xba I enzyme, obtains fragment F2A-L; And then with Nsi I endonuclease bamhi F2A-L, obtaining 5 ' end is that Nsi I restriction enzyme site and 3 ' end are the fragment F2A-L of Xba I restriction enzyme site; With BamH I and Nsi I double digestion chimeric heavy chain H, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the heavy chain fragment H in Nsi I site; Link to each other with fragment F2A-L through the T4DNA ligase enzyme, obtaining 5 ' end is that BamH I restriction enzyme site and 3 ' end are the chimeric antibody full length fragment H-F2A-L in Xba I site;
< 3>structure of slow virus expression plasmid pwpI/H-F2A-L
With the fragment H-F2A-L of purifying and recovering, mend flat two ends with DNA end-filling test kit; Cut slow virus expression plasmid pWPI with the PmeI enzyme simultaneously; Behind the SAP dephosphorylation, be connected with H-F2A-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-F2A-L, and carries out sequence verification, promptly gets;
Said each primer does
H-A：5’-CGC?GGA?TCC?GCC?ACC?ATG?GGA?TGG?AGC?TGT?ATC?ATC?CTC?T-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’，
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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| CN103045646A (en) * | 2012-12-27 | 2013-04-17 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant adeno-associated virus vector for co-expression of two independent anti-arthritis molecules TNFR-Fc and CTLA4-FasL, as well as construction method and application of recombinant adeno-associated virus vector |
| CN105420276A (en) * | 2015-12-17 | 2016-03-23 | 深圳精准医疗科技有限公司 | lentiviral expression vector, preparing method and application thereof, and preparing method of recombined lentivirus |
| CN105483158A (en) * | 2015-12-17 | 2016-04-13 | 深圳精准医疗科技有限公司 | Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus |
| CN106222194A (en) * | 2016-08-03 | 2016-12-14 | 深圳麦客思鱼生物科技发展有限公司 | The plant source recombinant humanized shellfish of a kind of optimization cuts down preparation method and the medical applications of monoclonal antibody |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045646A (en) * | 2012-12-27 | 2013-04-17 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant adeno-associated virus vector for co-expression of two independent anti-arthritis molecules TNFR-Fc and CTLA4-FasL, as well as construction method and application of recombinant adeno-associated virus vector |
| CN103045646B (en) * | 2012-12-27 | 2015-02-25 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant adeno-associated virus vector for co-expression of two independent anti-arthritis molecules TNFR-Fc and CTLA4-FasL, as well as construction method and application of recombinant adeno-associated virus vector |
| CN105420276A (en) * | 2015-12-17 | 2016-03-23 | 深圳精准医疗科技有限公司 | lentiviral expression vector, preparing method and application thereof, and preparing method of recombined lentivirus |
| CN105483158A (en) * | 2015-12-17 | 2016-04-13 | 深圳精准医疗科技有限公司 | Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus |
| CN106222194A (en) * | 2016-08-03 | 2016-12-14 | 深圳麦客思鱼生物科技发展有限公司 | The plant source recombinant humanized shellfish of a kind of optimization cuts down preparation method and the medical applications of monoclonal antibody |
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