CN100391980C - Antitumour multitarget composite antigen, its coding gene and application - Google Patents
Antitumour multitarget composite antigen, its coding gene and application Download PDFInfo
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- CN100391980C CN100391980C CNB2006100890992A CN200610089099A CN100391980C CN 100391980 C CN100391980 C CN 100391980C CN B2006100890992 A CNB2006100890992 A CN B2006100890992A CN 200610089099 A CN200610089099 A CN 200610089099A CN 100391980 C CN100391980 C CN 100391980C
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Abstract
The present invention discloses an antitumor multitarget point compound antigen, its coding gene and application. Said invention is aimed at providing a kind of multitarget point compound antigen, its coding gene and application in the preparation of vaccine for preventing and/or curing tumor. Said antigen has the amino acid residue sequence of SEQ ID No.1 in sequence table, and its coding gene has DNA sequence of SEQ ID No.2 in sequence table. The invented multitarget point compound antigen and its coding gene have the obvious tumor-inhibiting effect.
Description
Technical field
The present invention relates to antigen and encoding gene thereof and application, particularly relate to a kind of multitarget composite antigen and encoding gene thereof and its application in preventing and/or treating property of preparation tumor vaccine and medicine.
Background technology
Malignant tumour is one of most important killer who threatens human health.The vascularization of the generation of tumour, development and transfer and tumour is closely related, and concerning the postoperative patient of radical operation, 5 years recurrence rates of patient of no capillary blood vessel invasion and attack are 11%, and the 5 years recurrence rates of patient that have capillary blood vessel invasion and attack are up to 50%.Because it is identical that the tumor area blood vessel has, therefore anti-angiogenic formation treatment is applicable to multiple noumenal tumour.With the blood vessel is target spot, inhibition vascularization, thereby suppresses tumor proliferation and shift to have become a new oncotherapy approach.
Studies show that, vascular endothelial growth factor (vascular endothelialgrowth factor is not only depended in the formation of blood vessel, VEGF) and acceptor (vascular endothelial growth factor receptors, VEGF-R), also be subjected to simultaneously extracellular matrix (extracellular matrix, ECM) influence [the Kerbel RS.A cancer therapy resistant to resistance[J] .Nature of protein receptor-integration plain (integrin), 1997,390 (6658): 335-336.Ellis LM..Angiogenesis and its role incolorectal tumor and metastasis formation[J] .Semin Oncol, 2004,31 (6): 3-9.].Wherein, VEGF-E, VEGF-R
2With α V β
3The closest with the relation that tumor vessel forms, can be used as and suppress the direct target that pathologic vessels forms.
VEGF is that a kind of silk of endothelial cell specific splits the source, and induction of vascular generates in vivo, is to act on one of the strongest angiogenesis factor.Have now found that VEGF family comprises VEGF-A, B, C, D, E and placenta growth factor (placentagrowth factor, PIGF), VEGF-E high expression level [Veikkola T in pathologic vessels only wherein, AlitaloK.VEGFs, receptors and angiogenesis[J] .CancerBiology, 1999,9 (3): 211-220.].Experiment confirm is arranged, use the monoclonal antibody of anti-VEGF and can seal excretory VEGF, the signal transduction of blocking-up vascularization process, thereby suppress growth of tumor and transfer [Pidgeon GP, Barr MP, Harmey JH, et al.Vascular endothel ial growth factor (VEGF) upregulates BCL-2and inhibitsapoptosis in human and murine mammary adenocarcinoma cells[J] .Br J Cancer, 2001,85 (2): 273-278.Lichtenbeld HC, Ferarra N, Jain RK, et al.Effectof local anti-VEGF antibody treatment on tumor microvessel permeability[J] .Microvasc Res, 1999,57 (3): 357-362.].Anti-VEGF-E monoclonal anti is embodied in that to be in the III phase clinical.VEGF-E separates from the sheep of virus infected group, is a kind of secretor type dimer glycoprotein, both can be secreted into the extracellular, also can be incorporated into cell surface or extracellular matrix, has best bioavailability and biotic potential.Each monomer contains 8 distinctive cysteine residues, and these halfcystines form 3 ring texture: loop-1 ,-2 and-3 by disulfide linkage, constitute its main functional area.
The EGF receptor family has been found 5 kinds at present, wherein VEGF-R
1(Flt-1), VEGF-R
2(KDR/Flk-1) and VEGF-R
3(Flt-4) (other two kinds is Npn (neuropillin)-1 and-2 for receptortyrosine kinases, RTKs) superfamily, and they bring into play key effect in endothelial cell differentiation and vascularization to belong to the tyrosine protein kinase acceptor.VEGF-R
2High expression level in the embryonic blood vessel endotheliocyte, and in ripe blood vessel, express descending, illustrate it promote blood vessel to take place and regeneration in work.VEGF-R
2The extracellular region territory comprise seven immunoglobulin-like zones, Tyrosylprotein kinase zone and one and stride diaphragm area, wherein the ligand binding domain of high-affinity is formed in second and the 3rd immunoglobulin-like zone.
In recent years, more and more perfect to the research of vascularization mechanism.Discover that endothelial cell growth factor (ECGF) and acceptor thereof are not only depended in tumor vascular formation, also be subjected to extracellular matrix (extracellular matrix, ECM) protein receptor-integrin alpha V β
3Influence [Stromblad S, Becker JC, Yebra M, et al.Suppression of p53activity and p21WAF1/CIP1expression by vascular cellintegrin alphaVbeta3during angiogenesis[J] .J Clin Invest, 1996,98 (2): 426-433.].The neonatal blood vessels endotheliocyte is expressed high-caliber α V β in the tumor tissues
3, do not express or some organizes utmost point low expression level and have in adult's normal blood vessels endotheliocyte.It can cause the activity of MMP-2 matrix degradation at endothelial cell surface, and suppresses the p53 and the proteic activity of the inductive p21 of institute thereof of endotheliocyte, thereby shows anti-apoptosis effect.A large amount of experiments show, use integrin alpha V β in the animal body
3Antagonist can cause participating in generating the apoptosis of the endotheliocyte of blood vessel; Anti-α V β
3Antibody or α V β
3The antagonist new vessel that can destroy quail embryo, mouse retina, rabbit corneal generate; Use α V β in the tumor model
3Antagonist not only stoped the relevant vasculogenesis of tumour, and can cause shrinking back of tumour.α V β
3Be by α V chain and β
3The non-covalent transmembrane glycoprotein in conjunction with a heterodimer that constitutes of chain is an energy and the acceptor of ECM broad incorporation, and the ECM albumen of the every RGD of having sequence (Arg-Gly-Asp) all can combine with it.
Summary of the invention
The purpose of this invention is to provide a kind of to the inhibited multitarget composite antigen of tumour.
Multitarget composite antigen provided by the present invention, called after ERV has SEQ ID № in the sequence table: 1 amino acid residue sequence.
SEQ ID № in the sequence table: 1 is made up of 404 amino-acid residues, from amino (N) end 1-96 position for being the antigen sequence of target spot with blue tongue virus VEGF-E, wherein, from aminoterminal 23-30 position is the amino acid residue sequence of Loop1, from aminoterminal 46-51 position is the amino acid residue sequence of Loop2, is the amino acid residue sequence of Loop3 from aminoterminal 68-73 position; From aminoterminal 97-238 position is with Africa xenopus α V β
3Antigen sequence for target spot; From aminoterminal 239-404 position is with VEGF-R
2Being the antigen sequence of target spot, wherein, is the VEGF-R that derives from quail from aminoterminal 243-293 position
2The amino acid residue sequence in second immunoglobulin-like zone is the VEGF-R that derives from mouse from aminoterminal 339-400 position
2The amino acid residue sequence in the 3rd immunoglobulin-like zone is the aminoacid sequence that derives from people's joining region from aminoterminal 294-338 position.
The gene of above-mentioned multitarget composite antigen ERV of encoding also belongs to protection scope of the present invention, can have SEQ ID № in the sequence table: 2 dna sequence dna.
SEQ ID № in the sequence table: 2 by 1212 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 1-1212 bit base.Wherein, be the antigen sequence of target spot from 5 ' end 1-288 bit base coding with blue tongue virus VEGF-E, encode with Africa xenopus α V β from 5 ' end 289-714 bit base
3Be the antigen sequence of target spot, encode with VEGF-R from 5 ' end 715-1212 bit base
2Antigen sequence for target spot.
Contain multitarget composite antigen ERV expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
Increase arbitrary segmental primer in the described antigen gene to also within protection scope of the present invention.
Also belong to protection scope of the present invention with the antibody of above-mentioned multitarget composite antigen ERV specific combination, described antibody comprises monoclonal antibody and polyclonal antibody, all can prepare according to ordinary method.
Another object of the present invention provides a kind of method of expressing above-mentioned multitarget composite antigen ERV.
The method of the above-mentioned multitarget composite antigen ERV of expression provided by the present invention is that the recombinant expression vector that will contain above-mentioned multitarget composite antigen ERV gene imports host cell, expresses obtaining multitarget composite antigen ERV.
The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, as pGEX-4T-2, pET-3a, pET-30a, pET-28a, pET-28b or pET-28c, is preferably pGEX-4T-2.
Be the carrier that sets out with pGEX-4T-2, the recombinant expression vector that contains described vascular endothelial growth factor VEGF-E antigen gene of structure is pGEX-ERV.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Described intestinal bacteria can be E.coliBL21 (DE3), E.coli JM109, E.coliHB101 or E.coliTop10 etc.
Can adopt the conventional culture condition that makes up the used starting strain of engineering bacteria that engineering bacteria is cultivated, when described engineering bacteria is recombination bacillus coli, need to add the IPTG inductor, add IPTG concentration be 0.8-1.2mmol/L, be preferably 1mmol/L, inducing temperature is 35-39 ℃, is preferably 37 ℃, induction time is 2-4 hour, is preferably 3 hours.
Multitarget composite antigen ERV of the present invention can be used for preventing and/or treating property of preparation tumor vaccine, and the gene of coding multitarget composite antigen ERV can be used for preventing and/or treating property of preparation tumour dna vaccination.
Multitarget composite antigen ERV gene in described the preventing and/or treating property tumour dna vaccination can be present in the carrier for expression of eukaryon.
The carrier that sets out that is used for making up the carrier for expression of eukaryon that carries multitarget composite antigen ERV gene can be any one and can be preferably pCI-GPI at the expression vector of Mammals expression alien gene, and its physical map is seen Figure 18.
Be the carrier that sets out with pCI-GPI, the recombinant expression vector that carries multitarget composite antigen ERV gene of structure is pCI-ERV.
When needing, can also incorporate the gene of one or more cytokines such as granular leucocyte-macrophage colony stimulating factor (GM-CSF), interferon-(IFN-γ), interleukin-22 (IL-2), TGF-β 4 or protein in the vaccine with the vascular endothelial growth factor VEGF-E antigen preparation as the molecular immune adjuvant.
The invention provides multitarget composite antigen ERV and encoding gene thereof.With the eukaryon expression plasmid that contains multitarget composite antigen ERV gene of the present invention respectively as the immunogen immune mouse, the result compares with the control group of PBS and empty carrier, all produced the antibody of higher level in the antigen group mice serum, the T lymphocyte quantity of secretion of gamma-IFN and IL-4 obviously increases CD4 in the antigen group mouse boosting cell
+/ CD8
+Ratio also obviously raise, and the more single target spot antigen of multitarget composite antigen ERV of the present invention more can excite the humoral immune reaction of mouse; After immune mouse is accepted the attack of subcutaneous transplantation knurl, compare with the empty carrier control group with PBS, the one-tenth knurl time lengthening of antigen group mouse, tumor growth slowly and knurl volume-diminished, tumour inhibiting rate obviously improve, and the antigenic tumor killing effect of the more single target spot of multitarget composite antigen ERV of the present invention is more remarkable, and tumour inhibiting rate can reach 95%.Above-mentioned experimental result shows that multitarget composite antigen ERV of the present invention and encoding gene thereof have significant tumor killing effect, and has the expression amount height, easily purifying, with short production cycle, industrial scale is big, and the advantage that cost is low can be prepared into preventing and/or treating property tumour medicine and vaccine.The present invention has bigger practical significance and wide application prospect in medical science and field of biological pharmacy.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is for dividing the mode chart of five step pcr amplification VEGF-E target spot genes with the center die plates method
Fig. 2 is the agarose gel electrophoresis detected result of the VEGF-E target spot gene of pcr amplification
Fig. 3 is for dividing five step pcr amplification α V β with the center die plates method
3The mode chart of target spot gene
Fig. 4 is the α V β of pcr amplification
3The agarose gel electrophoresis detected result of target spot gene
Fig. 5 is for dividing five step pcr amplification α V β with the center die plates method
3The mode chart of target spot gene
Fig. 6 is the α V β of pcr amplification
3The agarose gel electrophoresis detected result of target spot gene
Fig. 7 is the agarose gel electrophoresis detected result of the multitarget composite antigen ERV gene of pcr amplification
Fig. 8 is the RT-PCR qualification result of stable transfected cells mRNA
Fig. 9 is the immunofluorescence detected result of stable transfected cells
Figure 10 is the Western Blotting qualification result of stable transfected cells
Figure 11 is the ELISPOT detected result of the IFN-γ and the IL-4 of immune mouse
Figure 12 is CD in the immune mouse
4 +And CD
8 +The cells were tested by flow cytometry result of T cell count ratio
Figure 13 is CD in the immune mouse
4 +And CD
8 +The histogram of T cell count ratio
Figure 14 attacks the back knurl cubing result who respectively organized immune mouse in 50 days for the subcutaneous transplantation knurl
Figure 15 attacks the back knurl remeasurement result who respectively organized immune mouse in 50 days for the subcutaneous transplantation knurl
Figure 16 for the B group be control group respectively organize immune mouse press down tumour rate statistics
Figure 17 attacks for the subcutaneous transplantation knurl and afterwards respectively organized the survival condition of immune mouse in 50 days, at body knurl volume and external knurl volume
Figure 18 is the physical map of carrier pCI-GPI
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.All primers synthesize and examining order is finished by Beijing three rich biological company limiteds.
The design of embodiment 1, multitarget composite antigen and the clone of encoding gene thereof
One, the design of multitarget composite antigen
Compare by literature search on a large scale (domestic and external) and sequence, obtained a series of VEGF-E, VEGF-R
2And α V β
3Functional region and these zones homology difference in the different plant species sequence, tumor therapeutic vaccine provides the candidate sequence in order to prepare efficiently.The core area of finally choosing blue tongue virus VEGF-E comprises the target spot of the polypeptide of being made up of 96 amino-acid residues of Loop-1, Loop-2 and Loop-3 as VEGF-E, it is SEQ ID № in the sequence table: certainly amino (N) end 1-96 amino acids residue of 1, wherein, from aminoterminal 23-30 position is the amino acid residue sequence of Loop1, from aminoterminal 46-51 position is the amino acid residue sequence of Loop2, is the amino acid residue sequence of Loop3 from aminoterminal 68-73 position; Choose Africa xenopus α V β
3Functional fragment in the total length comprises: from aminoterminal 118-131, and 126-129,217-231,211-221,109-118,99-118, the 209-220 position, the polypeptide that relates to 142 amino-acid residues compositions altogether is as α V β
3Target spot, i.e. SEQ ID № in the sequence table: 1 from aminoterminal 97-238 amino acids residue; Choose and comprise VEGF-R
2The polypeptide that extracellular region second and the 3rd immunoglobulin-like zone are made up of 166 amino-acid residues is as VEGF-R
2Target spot, i.e. SEQ ID № in the sequence table: 1 from aminoterminal 239-404 amino acids residue, wherein, be the VEGF-R that derives from quail from aminoterminal 243-293 position
2The amino acid residue sequence in second immunoglobulin-like zone is the VEGF-R that derives from mouse from aminoterminal 339-400 position
2The amino acid residue sequence in the 3rd immunoglobulin-like zone is the aminoacid sequence that derives from people's joining region from aminoterminal 294-338 position; At last with above-mentioned VEGF-E, VEGF-R
2With α V β
3Functional region couple together in turn, obtain the heterology multitarget composite antigen, called after ERV.
Two, the clone of multitarget composite antigen ERV gene
Derive the nucleotide sequence of this sequence of coding according to the aminoacid sequence of the multitarget composite antigen ERV of step 1 design, be the SEQ ID № in the sequence table: 2, by 1212 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 1-1212 bit base.Wherein, from 5 ' end 1-288 bit base coding blue tongue virus VEGF-E target sequence, from 5 ' end 289-714 bit base coding Africa xenopus α V β
3Target sequence is from 5 ' end 715-1212 bit base coding VEGF-R
2Target sequence.Synthesize multitarget composite antigen ERV gene with following method:
1, the clone of blue tongue virus VEGF-E target spot gene
According to blue tongue virus VEGF-E target spot gene order, be SEQ ID № in the sequence table: 2 from 5 ' end 1-288 position nucleotide sequence design pcr amplification primer, and add restriction enzyme Xho I and EcoR V recognition site respectively at primers F 5, R5 two ends, primer sequence is as follows:
F1?5’-AACTGTTAAACGATGCGGCGGTTGCTGTAATGACGACGGTCAAA-3’
R1?5’-TTGTATTTCTTGTTTCAACCGCTGTACATATTTGACCGTCATCG-3’；
F2?5’-ACTAACCTACAATATAATCCCCGGTGCGTAACTGTTAAACGATG-3’
R2?5’-AGACACGCCGGTTACTGAAACTGTTACAGTTGTATTTCTTGTTT-3’；
F3?5’-TTTATTTGGGAGAAGAATATCCAGAAAGCACTAACCTACAATAT-3’
R3?5’-GATACACCACTATTAGTACCAGACGAACTAGACACGCCGGTTAC-3’；
F4?5’-AAGTGGTTGTAAACCTAGAGATACTGTAGTATATTTGGGAGAAG-3’
R4?5’-CTGTAACACTTGTTCTTTGAAGGTTAGTAGATACACCACTATTA-3’；
F5 5 '-GC
CTCGAGTGGATGCGTACACTAGACAAAAGTGGTTGTAAACC-3 ' (band underscore base is a restriction enzyme Xho I recognition site)
R5 5 '-GC
GATATCACAATCGCACTTTGTGTGTTCTGTAACACTTGTTC-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).
Adopt the center die plates method to divide five step pcr amplification goal gene, synthesis model figure sees that (F: upstream primer, R: downstream primer), concrete grammar is as follows: the first step amplification system is Fig. 1: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs mixture (2.5mM) 4 μ l, each 1 μ l of synthetic primer (20 μ M) R1, F1, Taq archaeal dna polymerase (5U/ μ l) 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.
Four steps amplification is subsequently carried out as template after all diluting 50 times with previous step PCR product.Amplification system is the same, just the second step pcr amplification is selected primer R2 and F2 for use, and the 3rd step was selected primer R3 and F3 for use, and the 4th step was selected primer R4 and F4 for use, the 5th step was selected R5-1, F5-1 and R5-2, F5-2 respectively for use, and last takes turns the purifying of PCR multiplication of system to 100 μ l in order to the PCR product.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ extend 30s, totally 30 circulations; Last 72 ℃ are continued to extend 10min.Behind five pcr amplifications, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 2; Swimming lane 1.PCR end product (288bp); Swimming lane 2-5. substep PCR product), the result has obtained the gene fragment (arrow indication fragment among Fig. 2) that length is about 288bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier (TaKaRa company), check order to containing the pulsating recombinant vectors of recovery, result's segment that increases has SEQ ID № in the sequence table: 2 from 5 ' end 1-288 position nucleotide sequence, and extension increasing sequence is correct.
2, Africa xenopus α V β
3The clone of target spot gene
According to Africa xenopus α V β
3Target spot gene order, i.e. SEQ ID № in the sequence table: 2 from 5 ' end 289-714 position nucleotide sequence design pcr amplification primer, and add restriction enzyme XhoI and EcoR V recognition site respectively at primers F 51, R51 two ends, primer sequence is as follows:
Leading portion F11 5 '-ATGATTTAATTAAGATCCAAACCCTGGGCACTAGTTTGTCAGAGA-3 '
Leading portion R11 5 '-ATCCGCAGGTTACTAGTTAGCCGGCGCATCCTCTCTGACAAACTA-3 ';
Leading portion F21 5 '-ACCTTATGGACCTTTCCTATTCCATGAAGGATGATTTAATTAAGA-3 '
Leading portion R21 5 '-ATCGGCTTGTCAACGAATGCACCAAATCCAATCCGCAGGTTACTA-3 ';
Leading portion F31 5 '-AAGTAGAAGACTATCCTGTGGACATATACTACCTTATGGACCTTT-3 '
Leading portion R31 5 '-TCAGGAGGTGACATGAACATGTACGGTGACATCGGCTTGTCAACG-3 ';
Leading portion F415 '-GTCTTCAACCTACAGGTGAGGCAAGTAGAAGACTATC-3 '
Leading portion R41 5 '-ATAGCATGGGTTCTTGATGACTTCAGGAGGTGACATG-3 ';
Back segment F11 5 '-GCACGTCTTGACTTTGACAGAGGAAGTCTTGCTGTTTAACGAGGAGGT-3 '
Back segment R11 5 '-GAGAATCCCGGTTTCGGGAGACCTTCTGTTTCTGGACCTCCTCGTTAA-3 ';
Back segment F21 5 '-TCAATACCGAGTGCATGCCGACATTCGGATATAAGCACGTCTTGACTT-3 '
Back segment R21 5 '-GCTGCCTGTAGCACCGCATCAAATCCACCCTCTGGAGAATCCCGGTTT-3 ';
Back segment F31 5 '-CCTGAAGTCATCAAGAATCCATGCTATGAATTCAATACCGAGTGC-3 '
Back segment R31 5 '-ATTTCTCCAGCCAATCTTCTCGTCGCATACTGCTGCCTGTAGCAC-3 ';
F51 5 '-GC
CTCGAGGTCTTCAACCTACAGGTGAGGCAAG-3 ' (band underscore base is a restriction enzyme Xho I recognition site)
R51 5 '-GC
GATATCATTTCTCCAGCCAATCTTCTCGTCG-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).
Adopt the center die plates method to divide five step pcr amplification goal gene, synthesis model figure sees Fig. 3 (F: upstream primer, the R downstream primer), full gene is divided into forward and backward two sections, two fragments all obtain goal gene with four step PCR synthesis methods, utilize PCR method that the rear and front end is stitched together then, concrete grammar is as follows: the first step amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of primer R1, F1 (20 μ M), TaqDNA polysaccharase (5U/ μ l) 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.
Second step, the 3rd step and the amplification of the 4th step are carried out as template after all diluting 50 times with previous step PCR product.Amplification system is the same, and just primer second goes on foot pcr amplification and selects primer and F2 for use, and the 3rd step was selected primer R3 and F3 for use, and the 4th step was selected primer R4 and F4 for use.The PCR reaction conditions is: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations.Two sections goal gene of gained behind four pcr amplifications, called after leading portion and back segment respectively.
Final step pcr amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of synthetic primer (20 μ M) F5 and R5, Taq archaeal dna polymerase (5U/ μ l) 1 μ l, the leading portion 1 μ l after diluting 50 times, the back segment 1 μ l after diluting 50 times adds deionized water to 50 μ l.The PCR reaction conditions is with step 2 to four.Behind five pcr amplifications, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 4; Swimming lane 1-4. leading portion substep PCR product; Swimming lane 5. α V β
3PCR end product (426bp); Swimming lane 6-8. back segment substep PCR product), the result has obtained the gene fragment (arrow indication fragment among Fig. 4) that length is about 426bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier, check order to containing the pulsating recombinant vectors of recovery, result's segment that increases has SEQ ID № in the sequence table: 25 ' end 289-714 position nucleotide sequence, extension increasing sequence is correct.
3, VEGF-R
2The clone of target spot gene
According to VEGF-R
2The nucleotide sequence of target spot gene designs the pcr amplification primer, and adds restriction enzyme Xho I and EcoR V recognition site respectively at primers F 52, R52 two ends, and primer sequence is as follows:
Leading portion F12 5 '-ATTTCGTGGGATAATAAAAAAGGCTTCACTATACCCAGTCATCTAA-3 '
Leading portion R12 5 '-GCTTCACAGAAGACCATGCCTGCGTAGTTGATTAGATGACTGGGTA3 ';
Leading portion F22 5 '-AGAAAAGGTGTTCGTTCCTGACGGTAAATCCATTTCGTGGGATAAT-3 '
Leading portion R22 5 '-AATAGACTGGTAACTTTCATCATTAATTTTTGCTTCACAGAAGACC-3 ';
Leading portion F32 5 '-ATCTCAATGTATCTCTACATGCGAAATATCCAGAAAAGGTGTTCGT-3 '
Leading portion R32 5 '-TCCTATACCCTACAACGACAACTATGTACATAATAGACTGGTAACT-3 ';
Leading portion F42 5 '-GTGGTGATTCCATGCCTTGGAACTGTGTCAAATCTCAATGTATCTC-3 '
Leading portion R42 5 '-CCATGAGACGGACTCAGAACCACATCATAAATCCTATACCCTACAA-3 ';
Back segment F12 5 '-GGCTTGATTTCACCTGGCACTCTCCACCTTCAAAGTCTCATCATAA-3 '
Back segment R12 5 '-AAAGGGTTTCACATCCCGGTTTACAATCTTCTTATGATGAGACTTT-3 ';
Back segment F22 5 '-AATTGTACAGCGAGAACAGAGCTCAATGTGGGGCTTGATTTCACCT-3 '
Back segment R22 5 '-TGCTCAAAAACATCTTCGCCACAGTCCCAGGAAAGGGTTTCACATC-3 ';
Back segment F32 5 '-TGAACTATCTGTTGGAGAAAAGCTTGTCTTAAATTGTACAGCGAGA-3 '
Back segment R32 5 '-TCACTCTTGGTCACACTTTCTATTGTCAAGGTGCTCAAAAACATCT-3 ';
Back segment F42 5 '-ATGATGTGGTTCTGAGTCCGTCTCATGGAATTGAACTATCTGTTGG-3 '
Back segment R42 5 '-ACTGGATGCTGCACAGGTGTATTCCCCTTGGTCACCTTGGTCACA-3 ';
F52 5 '-GC
CTCGAGGTGGTGATTCCATGCCTTGGAACTG3 ' (band underscore base is a restriction enzyme Xho I recognition site)
R52 5 '-GC
GATATCACTGGATGCTGCACAGGTGTATTCC-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).
Adopt the center die plates method to divide five step pcr amplification goal gene, synthesis model figure sees Fig. 5, full gene is divided into forward and backward two sections, two fragments all obtain goal gene with four step PCR synthesis methods, utilize PCR method that the rear and front end is stitched together then, concrete grammar is as follows: the first step amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of upstream and downstream primer (20 μ M), Taq archaeal dna polymerase (5U/ μ l) 1 μ l adds deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.
Second step, the 3rd step and the amplification of the 4th step are carried out as template after all diluting 50 times with previous step PCR product.Amplification system is the same, and just primer second goes on foot pcr amplification and selects primer and F2 for use, and the 3rd step was selected primer R3 and F3 for use, and the 4th step was selected primer R4 and F4 for use.The PCR reaction conditions is: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations.Two sections goal gene of gained behind four pcr amplifications, called after leading portion and back segment respectively.
Final step pcr amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of synthetic primer (20 μ M) F5 and R5, Taq archaeal dna polymerase (5U/ μ l) 1 μ l, the leading portion 1 μ l after diluting 50 times, the back segment 1 μ l after diluting 50 times adds deionized water to 50 μ l.The PCR reaction conditions is with step 2 to four.Behind five pcr amplifications, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 6; Swimming lane 1-4. leading portion substep PCR product; Swimming lane 5 and 6.PCR end product (498bp); Swimming lane 7-10. back segment substep PCR product), the result has obtained the gene fragment (arrow indication fragment among Fig. 6) that length is about 498bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier, check order to containing the pulsating recombinant vectors of recovery, result's segment that increases has SEQ ID № in the sequence table: 2 from 5 ' end 715-1212 position nucleotide sequence, extension increasing sequence is correct.
3, the clone of multitarget composite antigen ERV gene
Utilize round pcr, divided for five steps above-mentioned VEGF-E, VEGF-R2 and α V β
3The target spot gene couples together synthetic ERV fusion gene, according to the nucleotide sequence design pcr amplification primer of ERV gene, and adds restriction enzyme Xho I and EcoR V recognition site respectively at primers F 13, R33 two ends, and primer sequence is as follows:
F13 5 '-GC
CTCGAGTGGATGCGTACACTAGACAA-3 ' (band underscore base is a restriction enzyme Xho I recognition site)
R13?5’-CTGTAGGTTGAAGACACAATCACACTTTGT-3’；
F23?5’-ACAAAGTGTGATTGTGTCTTCAACCTACAG-3’
R23?5’-ATGGAATCACCACCAAGACGTGCTTATA-3’；
F33?5’-TATAAGCACGTCTTGGTGGTGATTCCAT-3’
R33 5 '-GC
GATATCACTGGATGCTGCACAGGTGTATTCC-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).
First three step is with VEGF-E, VEGF-R
2With α V β
3The target spot gene adds catenation sequence respectively, in order to connecting.The pcr amplification system is: 10 * PCR damping fluid, 5 μ l, MgCl
2(2.5mM) 5 μ l, dNTPs (2.5mM) 4 μ l, each 1 μ l of primer (20 μ M) R13, F13 (or R23/F23 or R33/F33), Taq archaeal dna polymerase (5U/ μ l) 1 μ l, VEGF-E target spot gene (or VEGF-R
2Target spot gene or α V β 3 target spot genes) 1 μ l, add deionized water to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 30s then, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 5 circulations; Last 72 ℃ are continued to extend 10min.
The 4th step was with VEGF-E and α V β
3The target spot gene couples together, with VEGF-E and the α V β that is added with catenation sequence
3Simultaneously as template, carry out pcr amplification under the guiding of primer R23 and F13 after the PCR product of target spot gene dilutes 50 times, the pcr amplification system is identical with first three step with reaction conditions.
The 5th step was to be connected to form the ERV fusion gene, to be added with the VEGF-R of catenation sequence
2Simultaneously as template, carry out pcr amplification under the guiding of primer R33 and F13 after target spot gene and the 4th step PCR product dilute 50 times, the pcr amplification system is identical with first three step with reaction conditions.Behind five pcr amplifications, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 7; Swimming lane 1.ERV fusion gene; Swimming lane 2. is added with the α V β of catenation sequence
3The target spot gene; Swimming lane 3. is added with the VEGF-E target spot gene of catenation sequence; Swimming lane 4. is added with the VEGF-R of catenation sequence
2The target spot gene), the result has obtained the target gene fragment (seeing swimming lane 1) that length is about 1212bp through pcr amplification, conforms to expected results." the quick glue of PCR segment reclaims test kit " and reference reagent box specification sheets with vast Tyke, Beijing biological gene technology limited liability company reclaim and this purpose fragment of purifying, it is cloned in the pMD18-T carrier, check order to containing the pulsating recombinant vectors of recovery, result's segment that increases has SEQ ID № in the sequence table: 2 nucleotide sequence, extension increasing sequence is correct.
The structure and the evaluation thereof of embodiment 2, multitarget composite antigen ERV gene eukaryotic expression vector
One, the structure of multitarget composite antigen ERV gene eukaryotic expression vector
After the multitarget composite antigen ERV gene of embodiment 1 amplification carried out double digestion with restriction enzyme Xho I and EcoR V, form with on the eukaryon expression plasmid pCI-neo+ basis of carrier pCI-GPI[in Promega company of same enzyme double digestion, making up.Mainly be between former multiple clone site Nhe I of pCI-neo+ and Xba I restriction enzyme site, to insert the universal sequence structure to form.The gene order of universal sequence is 5 '-NheI-signal peptide-Xho I-EcoRV-EcoR I-IgG Fc-GPI-Xba I-3 ', and the foreign immunologic molecular gene can select proper restriction site to insert between Nhe I-EcoR I.PCI-GPI is except that containing human IgG Fc section (GenBank number: Z17370) and glycosylation phosphatidylinositols GPI (GenBank number: XM676434) the encoding sequence, the main skeleton structure that also contains the pCI carrier, as the CMV early promoter, chimeric intron, the Neo selection markers, SV40 enhanser and ammonia benzyl resistant gene Ampr+ etc., physical map is seen Figure 18] connect 12-24 hour down at 16 ℃, linked system is: 10 * T4DNA connects damping fluid 1 μ l, T4DNA ligase enzyme (12u/ μ l) 1 μ l, ERV gene PCR purified product 4 μ l, pCI-GPI carrier 1 μ l adds sterilization distilled water to 10 μ l.To connect product transformed into escherichia coli DH5 α competent cell, transformant be coated the enterprising row filter of LB resistant panel that contains the 100mg/mL penbritin.Single bacterium colony of growing on resistant panel of picking carries out bacterium colony PCR and identifies then: single colony inoculation is contained 100mg/mL penbritin (Amp in 3mL
+) the LB liquid nutrient medium in enlarged culturing 3h.After cultivating end, getting 1 μ l bacterium liquid dilutes with 100 μ l water, boil 10min, the centrifugal 3min of 12000rpm gets supernatant and does template, carries out pcr amplification under the guiding of primer R33 and F13,25 μ lPCR reaction systems are: supernatant 5 μ l, each 1 μ l of primers F 5 and R5,10 * PCR damping fluid, 2.5 μ l, MgCl
22.5 μ l, dNTPs 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l adds sterilization distilled water to 25 μ l.PCR reaction conditions: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ were extended 10 minutes.After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, can obtain the positive recon that length is about the dna fragmentation of 1212bp through pcr amplification.The plasmid of the positive recombinant that extraction identifies through PCR is done further to identify with sequence measurement, and sequencing result shows that the sequence of goal gene and the position in carrier are all correct, has obtained the carrier for expression of eukaryon of ERV gene, called after pCI-ERV.Simultaneously with VEGF-E, the α V β of same procedure with embodiment 1 amplification
3And VEGF-R
2The target spot gene fragment also connects into respectively among the carrier pCI-GPI, obtains carrier for expression of eukaryon separately, respectively called after pCI-VEGFE, pCI-α V β
3And pCI-VEGFR
2
Two, the evaluation of eukaryon expression plasmid
1, transient transfection RenCa cell
Carry carrier for expression of eukaryon pCI-ERV, pCI-VEGFE, pCI-α V β respectively with what step 1 obtained
3And pCI-VEGFR
2The positive colony bacterium be inoculated in the LB liquid nutrient medium, cultivated 12-24 hour down at 37 ℃, extract test kit in a small amount and, use the transfection reagent of QIAGEN company then with the plasmid of QIAGEN company with reference to product description extraction plasmid
Transfection Reagent and with reference to product description with pCI-ERV, pCI-VEGFE, pCI-α V β
3And pCI-VEGFR
2Plasmid transfection renal carcinoma cell line RenCa is contrast with pCI-neo (Promega company).
2, the screening of stable transfected cells
Transfectional cell is cultivated about 48 hours when cell density reaches 50-70% and merges, abandon nutrient solution, use RPMI 1640 substratum (Gibco company) that contain G-418 (400 μ g/mL) instead and carry out the resistant cell screening and culturing, the RenCa cell with untransfected compares simultaneously.When control cells was almost all dead, G-418 concentration can be reduced to 200 μ g/mL to keep the screening effect.After two weeks, as seen have resistance clone to form, treat that it increases gradually after, with the pancreatin of 0.25% (mass percentage concentration) cell dissociation is got off, with limiting dilution assay picking mono-clonal and carry out enlarged culturing.
3, the evaluation of stable transfected cells
1) RT-PCR of stable transfected cells mRNA identifies
Pancreatin with 0.25% is made single cell suspension with the monoclonal cell digestion of enlarged culturing, is collected in the glass centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant; Inhale fully after PBS gives a baby a bath on the third day after its birth time and abandon supernatant; Re-suspended cell and counting, the whole density that makes cell is 1 * 10
6/ mL; Get the 1mL cell suspension and add 1mL Trizol mixing, extract cell total rna, having the cell of pCI-ERV plasmid to carry out RT-PCR with primer R33 and F13 to transfection then identifies, there is the cell of pCI-VEGFE plasmid to carry out RT-PCR with primers F 5 and R5 to transfection and identifies transfection is had pCI-α V β with primers F 51 and R51
3The cell of plasmid carries out RT-PCR and identifies with primers F 52 and R52 transfection is had pCI-VEGFR
2The cell of plasmid carries out RT-PCR and identifies that qualification result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 8; Swimming lane 1.pCI-VEGFE; Swimming lane 2.pCI-VEGFR
2Swimming lane 3.pCI-α V β
3Swimming lane 4.pCI-ERV), the pCI-VEGFE plasmid has all obtained the fragment that length is about 288bp, pCI-α V β through amplification
3Plasmid has all obtained the fragment that length is about 426bp, pCI-VEGFR through amplification
2Plasmid has all obtained the fragment that length is about 498bp through amplification, and the pCI-ERV plasmid has all obtained the fragment that length is about 1212bp through amplification, shows VEGF-E, α V β
3And VEGF-R
2Target spot gene and ERV gene have pCI-VEGFE, pCI-α V β in transfection respectively
3, pCI-VEGFR
2With on the mRNA level, express in the RenCa cell of pCI-ERV plasmid.
2) immunofluorescence of stable transfected cells detects
The stable transfected cells that step 1) has been identified is (with pCI-neo
+The RenCa cell of empty carrier transfection and the RenCa cell of untransfected are respectively as negative control and blank) trysinization with 0.25% is prepared into single cell suspension; After PBS washes 3 times, encapsulant (3%BSA) room temperature sealing 20-30 minute; Remove encapsulant, add fluorescein-labeled mouse-anti human IgG (available from China fir Golden Bridge Technology, Inc. in Beijing), 4 ℃ of lucifuges were hatched 12-24 hour; PBS washes 3 times, adjusts cell concn to 5 * 10
5/ mL, cell suspension drip on the slide glass, mountant (available from China fir Golden Bridge Technology, Inc. in Beijing) mounting.Detected result is (1.pCI-VEGFE transfectional cell as shown in Figure 9; 2.pCI-VEGFR
2Transfectional cell; 3.pCI-α V β
3Transfectional cell; 4.pCI-ERV transfectional cell; 5.pCI-neo
+Transfectional cell; 6.RenCa cell), show VEGF-E, α V β
3And VEGF-R
2Target spot gene and ERV gene obtain respectively to express in transfectional cell separately.
3) the Western Blotting of stable transfected cells identifies
With step 2) stable transfected cells identified is (with pCI-neo
+Transfectional cell and RenCa cell are contrast) use 0.25% trysinization, collecting cell, PBS washed twice; With the TBS that contains 1%Triton X-100 (50mMTris-Cl, pH7.6,150mM NaCl) lysing cell 30min; 4 ℃, the centrifugal 10min collection of 12000rpm supernatant; After testing sample carried out the 15%SDS-PAGE electrophoretic separation, albumen is transferred to nitrocellulose filter (voltage 15V shifted 1 hour) from polyacrylamide gel with half-dried transfer instrument; With the TBST that contains 5% skim-milk (TBS+0.05%Tween 20) room temperature sealing 1 hour; The mouse-anti human IgG monoclonal antibody (available from China fir Golden Bridge Technology, Inc. in Beijing, by 1: 500 dilution proportion) that adds the HRP mark was hatched 12-24 hour for 4 ℃; After the TBST washing, add the goat anti-mouse IgG (available from China fir Golden Bridge Technology, Inc. in Beijing, by 1: 2000 dilution proportion) of two anti-horseradish peroxidase-labeled, incubated at room 2 hours; After the TBST washing, with the ECL colour developing that exposes.Detected result is (swimming lane 1.pCI-VEGFE transfectional cell as shown in figure 10; Swimming lane 2.pCI-VEGFR2 transfectional cell; Swimming lane 3.pCI-α V β
3Transfectional cell; Swimming lane 4.pCI-ERV transfectional cell; Swimming lane 5.pCI-neo
+Swimming lane 6.RenCa cell), further proof shows VEGF-E, α V β
3And VEGF-R
2Target spot gene and ERV gene obtain respectively to express in transfectional cell separately.
One, the structure of multitarget composite antigen ERV gene prokaryotic carrier
The pCI-ERV and the pGEX-4T-2 (Pharmacia company) of purifying are carried out double digestion with restriction enzyme Sal I and Not I respectively, enzyme is cut product carry out the detection of 1.2% agarose gel electrophoresis, with " the PCR segment quick glue reclaim test kit " of vast Tyke, Beijing biological gene technology limited liability company and reference reagent box specification sheets reclaims and the endonuclease bamhi of the pCI-ERV of the pGEX-4T-2 of linearization of purifying and 1212bp, purpose fragment with purifying is connected 12-24 hour for 16 ℃ with linearizing pGEX-4T-2 carrier then, linked system is: 10 * ligase enzyme damping fluid, 1 μ l, T
4Dna ligase 1 μ l, goal gene 4 μ l, pGEX-4T-2 carrier 1 μ l is with distilled water postreaction system to 10 μ l.To connect product transformed into escherichia coli DH5 α competent cell, transformant is coated the enterprising row filter of LB resistant panel that contains the 100mg/mL penbritin, picking can obtain the positive recon that length is about the dna fragmentation of 1212bp through pcr amplification at the single bacterium colony that grows on the resistant panel and carry out bacterium colony PCR and identify under the guiding of primer R33 and F13 then.The plasmid of the positive recombinant that extraction identifies through PCR, do further to identify with sequence measurement, sequencing result shows that the sequence of goal gene and position in carrier are all correct, has obtained multitarget composite antigen ERV Prokaryotic Expression carrier, called after pGEX-ERV.
Two, a large amount of preparations of the acquisition of engineering bacteria and inclusion body
With pGEX-ERV transformed into escherichia coli BL21 (DE3) competent cell, filter out positive recombinant bacterial strain with the method identical with step 1, in 1: 100 ratio the positive bacterium of recombinating is seeded in the 1000mL LB liquid nutrient medium that contains the 100mg/mL penbritin and cultivates in a large number under 37 ℃ then, adding chemical inducer IPTG when being cultured to logarithmic phase is 1mmol/L to final concentration, and continuation was cultivated 12-24 hour under 37 ℃.After cultivating end, 4 ℃, the centrifugal 15min collection of 6000rpm thalline, (20mmol/L, pH8.0) resuspended bacterial sediment add N,O-Diacetylmuramidase (1mg/mL) in room temperature magnetic agitation 10min with 100mL TE damping fluid; The broken thalline of ultrasonic wave in the ice bath (30s/ opens, and 20s/ stops, 15 circulations); 4 ℃, the centrifugal 20min of 12000rpm are collected inclusion body precipitation and supernatant respectively.NaCl (TE preparation) with 1mol/L washs 1 time with the inclusion body precipitation, 4 ℃, the centrifugal 20min of 12000rpm are to remove the foreign protein in the inclusion body, wash 2 times with the TE damping fluid after abandoning supernatant, 4 ℃, the centrifugal 20min collecting precipitation of 12000rpm carry out purifying with following step again.
Three, the purifying of target protein
With the urea of 8mol/L dissolving inclusion body, add mercaptoethanol to 1% (body behind 20 ℃, the centrifugal 10min of 12000rpm
Long-pending percentage concentration); Go up sample after the TE damping fluid balance with Q-Sepharose Fast Flow anion-exchange column (available from Pharmacia company) usefulness 6mol/L urea; After going out to pass the peak with the TE buffer solution elution of 6mol/L urea,, collect relevant elution peak with NaCl (the TE damping fluid preparation of the 6mol/L urea) wash-out of different concns (concentration is 0.05M, 0.1M, 0.15M, 0.2M, 0.25M, 0.5M and 1M); Each peak sampling carrying out SDS-PAGE identifies, according to electrophoresis result, the target protein component is carried out S-Sepharose Fast Flow cationic exchange coloum again and SephardexG-50 post (available from Pharmacia company) carries out purifying, at last purified target protein being carried out SDS-PAGE detects, the result has obtained purified target protein, i.e. multitarget composite antigen ERV.
The antitumor activity of embodiment 4, multitarget composite antigen ERV detects
One, grouping of animal and immunity
6 the week age Balb/c mouse be divided into 7 groups, 9 every group.The A group is the blank group; The B group is the PBS control group; The C group is pCI-neo+ empty carrier group; The D-4 group is pCI-VEGFR
2Group; The E-2 group is pCI-α V β
3Group; The F-2 group is the pCI-VEGFE group; The G group is pCI-ERV group ((pCI-ERV)-R, (pCI-ERV)-V, (pCI-ERV)-E are parallel three groups).
With five kinds of plasmid DNA (pCI-neo, pCI-VEGFR
2(embodiment 2 makes up), pCI-α V β
3(embodiment 2 makes up), pCI-VEGFE (embodiment 2 makes up), pCI-ERV (embodiment 2 makes up)) press 1: 1 (m: mixed v) with transfection reagent LipofectAMINE respectively, vibration back room temperature was placed 15 minutes gently, obtain LipofectAMINE-plasmid dna complex compound, as immunity DNA.Quadriceps muscle of thigh muscle multi-point injection is adopted in immunity, 100 μ g/ only: at first inject 100 μ l, 0.25% bupivacaine pre-treatment inoculation position, behind the 72h at the same area initial immunity, respectively at the 3rd week and 6 all each booster immunizations 1 time.
Two, the ELISA of serum antibody titer detects
Every group of mouse got blood through the tail vein in each immunity back in 2 weeks, the centrifugal 10min of 3000rpm behind the room temperature placement 3h, get upper serum and detect antibody titers with the ELISA method, to verify antigenic humoral immune reaction, set up the negative control and the PBS blank of the preceding mouse serum of immunity simultaneously, the ELISA detection method is as follows:
It is 5 μ g/mL that the albumen of embodiment 3 preparation is diluted to final concentration with coating buffer, and bag is by elisa plate, and 4 ℃ left standstill 12-24 hour; PBST washing: get rid of the coating buffer of abandoning in the elisa plate hole, PBST washing 1 time; With 2% casein solution room temperature sealing 4h, discard confining liquid, pat dry elisa plate; To add each hole behind the immune mouse serum usefulness PBS doubling dilution, the 100ul/ hole, 37 ℃ leave standstill 30min; With the sheep anti-mouse igg (available from China fir Golden Bridge Technology, Inc. in Beijing) of PBST washing back adding HRP mark, 37 ℃ of reaction 20min; After the PBST washing, with the TMB 10min that develops the color, 2M sulfuric acid termination reaction; Detect the OD value of 450nm at last with SPECTRAIII enzyme connection instrument.The result judges: the OD value of testing sample is positive more than or equal to 2.1 (P/N 〉=2.1) with the ratio of the OD value of negative control, shows that the maximum antibody dilution of positive findings is the antibody titer of immune serum.Antibody titer detected result in the immune serum is as shown in table 1,
Antibody titer in table 1 immune serum
Annotate:
※With PBS, pCI-neo
+Compare with the isoantigen control group, this group mouse all produces specific antibody (P＜0.05)
*The twice booster immunization obviously antibody titer of enhancing immunity mice serum (P＜0.05) compared with the empty carrier control group with PBS, all produced the antibody of higher level in antigen group (D-4, E-2, F-2, the G group) mice serum, twice booster immunization be the antibody titer of enhancing immunity mice serum obviously, in addition, the more single target spot antigen of multitarget composite antigen ERV of the present invention more can excite the humoral immune reaction of mouse.
Three, the ELISPOT of the IFN-γ of immune mouse and IL-4 detects
1, the separation of immune mouse splenic lymphocyte
Every group of immune mouse got three, aseptic separation splenic lymphocyte, and method is: disconnected neck is put to death mouse, and the 5min that sterilizes in 75% ethanol puts into super clean bench immediately and becomes right arm reclining; Aseptic taking-up spleen is put in the plate (the 8mL serum-free RPMI1640 nutrient solution of interior dress precooling), rejects fat and reticular tissue as far as possible, pushes the spleen tissue gently with grinding rod on 200 order stainless (steel) wires; Splenocyte suspension 8mL slowly is equipped with in the transparent centrifuge tube of 4mL Ficoll lymphocyte separation medium (available from Pharmacia company) the centrifugal 20min of 2000rpm level along tube wall slow the adding; The careful tunica albuginea layer of drawing is washed (1000rpm, 10min) 2 times with the substratum of 10mL precooling, cell is resuspended in RPMI 1640 substratum that contain 20% foetal calf serum, adjusts cell concn to 1 * 10
7/ mL.
2, the ELISPOT of the IFN-γ of immune mouse and IL-4 detects
The splenic lymphocyte of the isolating immune mouse of step 1 is detected with the ELISPOT that Murine IFN γ and IL-4ELISPOT test kit and reference reagent box specification sheets carry out IFN-γ and IL-4, and detected result sees Table 2 and shown in Figure 11,
The ELISPOT of table 2 immune mouse IFN-γ and IL-4 detects
Annotate:
※Compare with B, C control group, ELISPOT detects this group mouse spot number and increases P＜0.05,
※ ※P＜0.01 is compared with PBS, empty carrier control group, the T lymphocyte quantity of secretion of gamma-IFN and IL-4 all obviously increases with the control group ratio in antigen group (D-4, E-2, F-2, the G group) mouse boosting cell, and the more single target spot antigen of multitarget composite antigen ERV of the present invention more can excite the humoral immune reaction of mouse.
Four, the T Lymphocyte Subsets Determination of immune mouse splenic lymphocyte
Get mouse boosting cell 1 * 10
6/ mL-2 * 10
6/ mL, PBS wash twice; The anti-CD that adds the PE mark respectively
4 +The anti-CD of mAb and FITC mark
8 +Each 20ul of mAb (available from the brilliant U.S. biological company limited in Beijing), 4 ℃ of lucifuge reaction 30min behind the mixing; After PBS washes twice, add 0.5mL fluorescence and preserve liquid (0.15mol/L PBS, 20g/L glucose, 10g/L formaldehyde and 1g/L NaN
3) re-suspended cell, with flow cytometry analysis T cell subsets, immune mouse CD
4 +And CD
8 +The cells were tested by flow cytometry result of T cell count ratio such as table 3 and Figure 12, shown in Figure 13.
The immune mouse CD of table 3 cells were tested by flow cytometry
4 +And CD
8 +The ratio of T cell count
Annotate:
※Compare this group mouse CD with B, C control group
4 +/ CD
8 +Ratio significantly increase P＜0.05,
*P＜0.01 is compared with the empty carrier control group with PBS, CD4 in antigen group (D-4, E-2, F-2, the G group) mouse boosting cell
+/ CD8
+Ratio obviously raise, and the more single target spot antigen of multitarget composite antigen ERV of the present invention more can excite the humoral immune reaction of mouse.
Five, detect the provide protection that immune mouse is attacked the subcutaneous transplantation knurl
A week remaining 6 immune mouses of each group being carried out the subcutaneous transplantation knurl after the last immunity attacks.The RenCa cell of results logarithmic phase, the preparation single cell suspension washes twice with physiological saline, adjusts cell concn to 1 * 10
7/ mL is 1 * 10 in mouse back right side subcutaneous vaccination 100 μ l cell suspensions
6/ only.After the subcutaneous transplantation knurl is attacked, observe the growing state of mice-transplanted tumor.Treat that tumor nodule forms back (can lay one's hand on and, the about 2-3mm of major diameter), write down the one-tenth knurl time of every group of mouse, observe the survival condition of mouse simultaneously.Attack back 50 days execution mouse in tumour cell, take pictures; With the vertical major diameter (a) and vertical minor axis (b) of vernier caliper measurement tumour, calculate the transplanted tumor volume according to formula; It is heavy to take by weighing knurl, calculates the inhibiting rate of tumour according to formula.
Each average one-tenth knurl time of organizing immune mouse subcutaneous transplantation knurl sees Table 4, the subcutaneous transplantation knurl is attacked the back survival condition of respectively organizing immune mouse in 50 days, see that at body knurl volume and external knurl volume Figure 17 from left to right is the mouse survival condition successively, show gross tumor volume at body, exsomatize and show gross tumor volume), wherein the survival rate statistics sees Table 5, the knurl cubing the results are shown in Table 6 and Figure 14, the knurl remeasurement the results are shown in Figure 15, each tumour rate statistics that presses down of organizing immune mouse sees Table 7, be that the histogram that presses down tumour rate statistics of respectively organizing immune mouse of control group is seen Figure 16 wherein with the B group
Table 4 is respectively organized the average one-tenth knurl time of immune mouse subcutaneous transplantation knurl
Annotate:
※With B, C control group relatively this group mouse become knurl prolongation of latency (P＜0.05)
Table 5 subcutaneous transplantation knurl is attacked the back survival condition of respectively organizing immune mouse in 50 days
Annotate:
※With B, C control group relatively this group mouse survival rate obviously increase (P＜0.05)
Table 6 subcutaneous transplantation knurl is attacked the back knurl volume of respectively organizing immune mouse in 50 days
Annotate:
※With B, C control group relatively this group mouse tumor volume obviously dwindle (P＜0.001)
Table 7 is respectively organized the restraining effect of immune mouse to tumour
Annotate:
※Compare with control group, this group mouse is to the restraining effect highly significant (P＜0.01) of tumour;
*After P＜0.001 immune mouse is accepted the attack of subcutaneous transplantation knurl, compare with the empty carrier control group with PBS, the one-tenth knurl time lengthening of antigen group (D-4, E-2, F-2, G group) mouse, tumor growth slowly and knurl volume-diminished, tumour inhibiting rate obviously improve, and the antigenic tumor killing effect of the more single target spot of multitarget composite antigen ERV of the present invention is more remarkable.
Sequence table
<160>2
<210>1
<211>404
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
Trp?Met?Arg?Thr?Leu?Asp?Lys?Ser?Gly?Cys?Lys?Pro?Arg?Asp?Thr?Val
1 5 10 15
Val?Tyr?Leu?Gly?Glu?Glu?Tyr?Pro?Glu?Ser?Thr?Asn?Leu?Gln?Tyr?Asn
20 25 30
Pro?Arg?Cys?Val?Thr?Val?Lys?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Asp
35 40 45
Gly?Gln?Ile?Cys?Thr?Ala?Val?Glu?Thr?Arg?Asn?Thr?Thr?Val?Thr?Val
50 55 60
Ser?Val?Thr?Gly?Val?Ser?Ser?Ser?Ser?Gly?Thr?Asn?Ser?Gly?Val?Ser
65 70 75 80
Thr?Asn?Leu?Gln?Arg?Ile?Ser?Val?Thr?Glu?His?Thr?Lys?Cys?Asp?Cys
85 90 95
Val?Phe?Asn?Leu?Gln?Val?Arg?Gln?Val?Glu?Asp?Tyr?Pro?Val?Asp?Ile
100 105 110
Tyr?Tyr?Leu?Met?Asp?Leu?Ser?Tyr?Ser?Met?Lys?Asp?Asp?Leu?Ile?Lys
115 120 125
Ile?Gln?Thr?Leu?Gly?Thr?Ser?Leu?Ser?Glu?Arg?Met?Arg?Arg?Leu?Thr
130 135 140
Ser?Asn?Leu?Arg?Ile?Gly?Phe?Gly?Ala?Phe?Val?Asp?Lys?Pro?Met?Ser
145 150 155 160
Pro?Tyr?Met?Phe?Met?Ser?Pro?Pro?Glu?Val?Ile?Lys?Asn?Pro?Cys?Tyr
165 170 175
Glu?Phe?Asn?Thr?Glu?Cys?Met?Pro?Thr?Phe?Gly?Tyr?Lys?His?Val?Leu
180 185 190
Thr?Leu?Thr?Glu?Glu?Val?Leu?Arg?Phe?Asn?Glu?Glu?Val?Gln?Lys?Gln
195 200 205
Lys?Val?Ser?Arg?Asn?Arg?Asp?Ser?Pro?Glu?Gly?Gly?Phe?Asp?Ala?Val
210 215 220
Leu?Gln?Ala?Ala?Val?Cys?Asp?Glu?Lys?Ile?Gly?Trp?Arg?Asn?Val?Val
225 230 235 240
Ile?Pro?Cys?Leu?Gly?Thr?Val?Ser?Asn?Leu?Asn?Val?Ser?Leu?His?Ala
245 250 255
Lys?Tyr?Pro?Glu?Lys?Val?Phe?Val?Pro?Asp?Gly?Lys?Ser?Ile?Ser?Trp
260 265 270
Asp?Asn?Lys?Lys?Gly?Phe?Thr?Ile?Pro?Ser?His?Leu?Ile?Asn?Tyr?Ala
275 280 285
Gly?Met?Val?Phe?Cys?Glu?Ala?Lys?Ile?Asn?Asp?Glu?Ser?Tyr?Gln?Ser
290 295 300
Ile?Met?Tyr?Ile?Val?Val?Val?Val?Gly?Tyr?Arg?Ile?Tyr?Asp?Val?Val
305 310 315 320
Leu?Ser?Pro?Ser?His?Gly?Ile?Glu?Leu?Ser?Val?Gly?Glu?Lys?Leu?Val
325 330 335
Leu?Asn?Cys?Thr?Ala?Arg?Thr?Glu?Leu?Asn?Val?Gly?Leu?Asp?Phe?Thr
340 345 350
Trp?His?Ser?Pro?Pro?Ser?Lys?Ser?His?His?Lys?Lys?Ile?Val?Asn?Arg
355 360 365
Asp?Val?Lys?Pro?Phe?Pro?Gly?Thr?Val?Ala?Lys?Met?Phe?Leu?Ser?Thr
370 375 380
Leu?Thr?Ile?Glu?Ser?Val?Thr?Lys?Ser?Asp?Gln?Gly?Glu?Tyr?Thr?Cys
385 390 395 400
Ala?Ala?Ser?Ser
<210>2
<211>1212
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
tggatgcgta?cactagacaa?aagtggttgt?aaacctagag?atactgttgt?ttatttggga 60
gaagaatatc?cagaaagcac?taacctacaa?tataatcccc?ggtgcgtaac?tgttaaacga 120
tgcggcggtt?gctgtaacga?tgacggtcaa?atatgtacag?cggttgaaac?aagaaataca 180
actgtaacag?tttcagtaac?cggcgtgtct?agttcgtctg?gtactaatag?tggtgtatct 240
actaaccttc?aaagaacaag?tgttacagaa?cacacaaagt?gcgattgtgt?cttcaaccta 300
caggtgaggc?aagtagaaga?ctatcctgtg?gatatctact?accttatgga?cctttcctat 360
tccatgaagg?atgatttaat?caagatccaa?accctgggca?ctagtttatc?agaaaggatg 420
cgccggctaa?ctagtaacct?gcggattgga?tttggtgcat?ttgttgacaa?gcctatgtca 480
ccgtacatgt?ttatgtcacc?tcctgaagtc?atcaaaaacc?cctgctatga?atttaacacc 540
gagtgcatgc?ccacttttgg?atataaacac?gtcttgactt?tgacagagga?agtcttgaga 600
tttaacgagg?aggtccagaa?acagaaggtc?tcccgaaacc?gggattctcc?agagggtgga 660
tttgatgcgg?tgctacaggc?agcagtatgc?gacgagaaga?ttggctggag?aaatgtggtg 720
attccatgcc?ttggaactgt?gtcaaatctc?aatgtatctc?tacatgcgaa?atatccagaa 780
aaggtgttcg?ttcctgacgg?taaatccatt?tcgtgggata?ataaaaaagg?cttcactata 840
cccagtcatc?taatcaacta?cgcaggcatg?gtcttctgtg?aagcaaaaat?taatgatgaa 900
agttaccagt?ctattatgta?catagttgtc?gttgtagggt?ataggattta?tgatgtggtt 960
ctgagtccgt?ctcatggaat?tgaactatct?gttggagaaa?agcttgtctt?aaattgtaca 1020
gcgagaacag?agctcaatgt?ggggcttgat?ttcacctggc?actctccacc?ttcaaagtct 1080
catcataaga?agattgtaaa?ccgggatgtg?aaaccctttc?ctgggactgt?ggcgaagatg 1140
tttttgagca?ccttgacaat?agaaagtgtg?accaagagtg?accaagggga?atacacctgt 1200
gcagcatcca?gt 1212
Claims (10)
1. multitarget composite antigen is by SEQ ID № in the sequence table: the amino acid residue sequence of 1 expression.
2. the gene of coding claim 1 described multitarget composite antigen.
3. gene according to claim 2 is characterized in that: described gene is by SEQ ID № in the sequence table: the dna sequence dna of 2 expressions.
4. contain claim 2 or 3 described expression carrier, transgenic cell line or host bacterium.
5. a method of expressing multitarget composite antigen is that the recombinant expression vector that will contain claim 2 or 3 described multitarget composite antigen genes imports host cell, expresses obtaining multitarget composite antigen.
6. method according to claim 5 is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector is pGEX-4T-2, pET-3a, pET-30a, pET-28a, pET-28b or pET-28c; Be the carrier that sets out with pGEX-4T-2, the recombinant expression vector that contains described multitarget composite antigen gene of structure is pGEX-ERV; Described host is E.coli BL21 (DE3), E.coli JM109, E.coli HB101 or E.coli Top10.
7. with the antibody of the described multitarget composite antigen specific combination of claim 1.
8. the application of the described multitarget composite antigen of claim 1 in preventing and/or treating property of preparation tumor vaccine or medicine.
9. claim 2 or 3 application of described multitarget composite antigen gene in preventing and/or treating property of preparation tumour dna vaccination or medicine.
10. application according to claim 9 is characterized in that: the multitarget composite antigen gene in the described dna vaccination is present in the carrier for expression of eukaryon; The described carrier for expression of eukaryon that carries the multitarget composite antigen gene is pCI-ERV.
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|---|---|---|---|
| CNB2006100890992A CN100391980C (en) | 2006-08-02 | 2006-08-02 | Antitumour multitarget composite antigen, its coding gene and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100890992A CN100391980C (en) | 2006-08-02 | 2006-08-02 | Antitumour multitarget composite antigen, its coding gene and application |
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| CN100391980C true CN100391980C (en) | 2008-06-04 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1727362A (en) * | 2004-07-30 | 2006-02-01 | 中国人民解放军第二军医大学 | Preparation and application of carcinoembryonic antigen positive tumor therapeutic vaccine |
| CN1730490A (en) * | 2005-08-03 | 2006-02-08 | 清华大学 | A kind of tumor related protein and its coding gene and application |
| CN1769298A (en) * | 2004-11-05 | 2006-05-10 | 中国人民解放军军事医学科学院生物工程研究所 | A fusion protein with anti-tumor effect and its coding gene and application |
-
2006
- 2006-08-02 CN CNB2006100890992A patent/CN100391980C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1727362A (en) * | 2004-07-30 | 2006-02-01 | 中国人民解放军第二军医大学 | Preparation and application of carcinoembryonic antigen positive tumor therapeutic vaccine |
| CN1769298A (en) * | 2004-11-05 | 2006-05-10 | 中国人民解放军军事医学科学院生物工程研究所 | A fusion protein with anti-tumor effect and its coding gene and application |
| CN1730490A (en) * | 2005-08-03 | 2006-02-08 | 清华大学 | A kind of tumor related protein and its coding gene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1903881A (en) | 2007-01-31 |
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