CN1903880B - Anti-tumor vascular endothelial growth factor VEGF-E antigen and its coding gene and application - Google Patents
Anti-tumor vascular endothelial growth factor VEGF-E antigen and its coding gene and application Download PDFInfo
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- CN1903880B CN1903880B CN200610089097A CN200610089097A CN1903880B CN 1903880 B CN1903880 B CN 1903880B CN 200610089097 A CN200610089097 A CN 200610089097A CN 200610089097 A CN200610089097 A CN 200610089097A CN 1903880 B CN1903880 B CN 1903880B
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Abstract
本发明公开了抗肿瘤血管内皮生长因子VEGF-E的抗原及其编码基因与应用。其目的是提供血管内皮生长因子VEGF-E的抗原及其编码基因与其在制备预防和/或治疗性肿瘤疫苗中的应用。该抗原具有序列表中SEQ ID №：1的氨基酸残基序列。其编码基因具有序列表中SEQ ID №：2的DNA序列。本发明的血管内皮生长因子VEGF-E抗原及其编码基因具有显著的抑瘤效果，且具有表达量高，易纯化，生产周期短，生产规模大，成本低的优点，可制备成预防和/或治疗性肿瘤疫苗及药物。本发明在医学和生物制药领域具有较大的实际意义和广阔的应用前景。The invention discloses an anti-tumor vascular endothelial growth factor VEGF-E antigen, its coding gene and application. The purpose is to provide the antigen of vascular endothelial growth factor VEGF-E and its encoding gene and its application in preparing preventive and/or therapeutic tumor vaccines. The antigen has the amino acid residue sequence of SEQ ID No.: 1 in the sequence listing. Its coding gene has the DNA sequence of SEQ ID No.: 2 in the sequence listing. The vascular endothelial growth factor VEGF-E antigen and its coding gene of the present invention have significant tumor-suppressing effects, and have the advantages of high expression, easy purification, short production cycle, large production scale, and low cost, and can be prepared as a preventive and/or Or therapeutic tumor vaccines and drugs. The invention has great practical significance and broad application prospects in the fields of medicine and biopharmaceuticals.
Description
Technical field
The present invention relates to vascular endothelial growth factor antigen and encoding gene thereof and application, particularly relate to a kind of antigen and encoding gene and its application in preventing and/or treating property of preparation tumor vaccine and medicine of vascular endothelial growth factor VEGF-E.
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.].In the various vascularization correlation factors, 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 endothelial growth factor (VEGF) upregulates BCL-2 and inhibitsapoptosis in human and murine mammary adenocarcinoma cells[J] .Br J Cancer, 2001,85 (2): 273-278.Licbtenbeld 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.
Summary of the invention
The purpose of this invention is to provide a kind of vascular endothelial growth factor VEGF-E antigen with antineoplastic vascular formation effect.
Vascular endothelial growth factor VEGF-E antigen provided by the present invention has SEQ ID № in the sequence table: 1 amino acid residue sequence.
SEQ ID № in the sequence table: 1 is made up of 96 amino-acid residues, from amino (N) end 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.
The gene of above-mentioned vascular endothelial growth factor VEGF-E antigen 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 288 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 1-288 bit base.Wherein, from 5 ' end 67-90 bit base coding Loop1, from 5 ' end 136-153 bit base coding Loop2, from 5 ' end 202-219 bit base coding Loop3.
Contain vascular endothelial growth factor VEGF-E antigen 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 vascular endothelial growth factor VEGF-E antigen 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 vascular endothelial growth factor VEGF-E antigen.
The method of the above-mentioned vascular endothelial growth factor VEGF-E antigen of expression provided by the present invention, be that the recombinant expression vector that will contain above-mentioned vascular endothelial growth factor VEGF-E antigen gene imports host cell, express obtaining vascular endothelial growth factor VEGF-E antigen.
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-VEGFE.
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.coli BL21 (DE3), E.coli JM109, E.coli HB101 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.
Vascular endothelial growth factor VEGF-E antigen of the present invention can be used for preventing and/or treating property of preparation tumor vaccine, and the gene of coding vascular endothelial growth factor VEGF-E antigen can be used for preventing and/or treating property of preparation tumour dna vaccination.
Vascular endothelial growth factor VEGF-E antigen 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 the vascular endothelial growth factor VEGF-E antigen 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 the vascular endothelial growth factor VEGF-E antigen gene of structure is pCI-VEGFE.
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 the antigen and the encoding gene thereof of vascular endothelial growth factor VEGF-E.With the eukaryon expression plasmid that contains respective section in vascular endothelial growth factor VEGF-E antigen gene of the present invention and the mouse respectively as the immunogen immune mouse, the result compares with PBS, empty carrier control group, 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 vascular endothelial growth factor VEGF-E antigen of the present invention more can excite the humoral immune reaction of mouse than the corresponding antigens in mouse source; After immune mouse is accepted the attack of subcutaneous transplantation knurl, compare with PBS, empty carrier control group, the one-tenth knurl time lengthening of antigen group mouse, tumor growth slowly and knurl volume-diminished, tumour inhibiting rate obviously improve, and vascular endothelial growth factor VEGF-E antigen of the present invention is more remarkable than the tumor killing effect of the corresponding antigens of mouse, and tumour inhibiting rate can reach more than 80%.Above-mentioned experimental result shows that vascular endothelial growth factor VEGF-E antigen 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 vascular endothelial growth factor VEGF-E antigen genes with the center die plates method
Fig. 2 is the agarose gel electrophoresis detected result of pcr amplification vascular endothelial growth factor VEGF-E antigen gene
Fig. 3 is the agarose gel electrophoresis detected result of the mVEGF gene fragment of pcr amplification
Fig. 4 is the RT-PCR qualification result of stable transfected cells mRNA
Fig. 5 is the immunofluorescence detected result of stable transfected cells
Fig. 6 is the Western Blotting qualification result of stable transfected cells
Fig. 7 is the structure schema of vascular endothelial growth factor VEGF-E antigen gene prokaryotic carrier
Fig. 8 is the agarose gel electrophoresis detected result of pCI-VEGFE and pGEX-4T-2 plasmid Sal I and Not I double digestion product
Fig. 9 is the SDS-PAGE detected result of the vascular endothelial growth factor VEGF-E antigen of prokaryotic expression and purifying
Figure 10 is the ELISPOT detected result of the IFN-γ and the IL-4 of immune mouse
Figure 11 is CD in the immune mouse
4 +And CD
8 +The cells were tested by flow cytometry result of T cell count ratio
Figure 12 is CD in the immune mouse
4 +And CD
8 +The histogram of T cell count ratio
Figure 13 is the average one-tenth knurl time of each group immune mouse subcutaneous transplantation knurl
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, vascular endothelial growth factor VEGF-E antigen and the clone of encoding gene thereof
One, the design of vascular endothelial growth factor VEGF-E antigen
Compare by literature search on a large scale (domestic and external) and sequence, obtained functional region and these zones homology difference in the different plant species sequence of a series of VEGF-E, 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 polypeptide of being made up of 96 amino-acid residues of loop-1, loop-2 and loop-3 as vascular endothelial growth factor VEGF-E antigen, be the SEQ ID № in the sequence table: 1, from amino (N) end 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.
Two, the clone of vascular endothelial growth factor VEGF-E antigen gene and mouse source corresponding gene thereof
Derive the nucleotide sequence of this sequence of coding according to the aminoacid sequence of the vascular endothelial growth factor VEGF-E antigen of step 1 design, be the SEQ ID № in the sequence table: 2, by 288 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 1-288 bit base.Wherein, from 5 ' end 67-90 bit base coding Loop1, from 5 ' end 136-153 bit base coding Loop2, from 5 ' end 202-219 bit base coding Loop3.Utilization utilizes the center die plates method to divide synthetic this gene of 5 steps, and in contrast, concrete grammar is as follows to clone corresponding gene segment (being called for short mVEGF) in the mouse simultaneously:
1, the clone of vascular endothelial growth factor VEGF-E antigen gene
Nucleotide sequence according to the vascular endothelial growth factor VEGF-E antigen gene of deriving designs the pcr amplification primer, and adds restriction enzyme Xho I and EcoR V recognition site respectively at primers F 5, R5 two ends, and 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(2.5mN) 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 step pcr amplifications carry out as template after all diluting 50 times with previous step PCR product subsequently.Amplification system is the same, and 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, and the 5th step was selected primer R5 and F5 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 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 (available from 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 nucleotide sequence, extension increasing sequence is correct.
2, the pulsating amplification of corresponding gene in the mouse
1) design primer
VEGF gene order (sequence number: NP-035827 according to mouse among the GeneBank, NM-011697) primer of design amplification respective regions (with this fragment called after mVEGF), and introduce the recognition site of restriction enzyme enzyme Xho I and EcoR V respectively at the two ends of upstream and downstream primer, primer sequence is as follows:
F6 (upstream primer): 5 '-GC
GCTAGCTGGATAGACGTTTATGCACGTGCC-3 ' (band underscore base is a restriction enzyme Xho I recognition site)
R6 (downstream primer): 5 '-GC
GATATCGCATTCACATTGGCTGTGTTCTTC-3 ' (band underscore base is a restriction enzyme EcoR V recognition site).
2) preparation mouse embryo tissue homogenate
Disconnected neck is put to death pregnant mouse, is soaked in 5min in 75% ethanol; Aseptic taking-up uterus and wherein embryo, placenta are rejected fat and reticular tissue as far as possible and are shredded with scissors; The tissue that shreds is put in adds the liquid nitrogen porphyrize in the mortar to Powdered, be collected in the glass homogenizer of ice precooling, add 1mL Trizol, homogenate is to transparent; Tissue homogenate changes in the aseptic RNase-free EP pipe standby rapidly.
3) the Trizol method is extracted total RNA of tissue homogenate
Add chloroform 200 μ l in the EP pipe of results tissue homogenate, put upside down mixing 15s immediately, room temperature leaves standstill 5min; 4 ℃/12000rpm, centrifugal 15min; Carefully upper phase is moved in the 1.5mL EP pipe of another new aseptic RNase-free, add the Virahol of equivalent, put upside down mixing, room temperature leaves standstill 10min; 4 ℃, the centrifugal 10min of 12000rpm; Abandon supernatant, add 75% ethanol 1mL, put upside down mixing, 4 ℃, the centrifugal 5min of 7500rpm abandon supernatant; Repeat thoroughly to abandon supernatant after the step, will precipitate seasoning, add DEPC water 10 μ l dissolution precipitations, be directly used in reverse transcription reaction.
4) reverse transcription reaction
Total RNA of the mouse embryo tissue that step 3) is extracted is a template, be primer with olig (dT) 20, and the SuperScript III RNase H-Reverse Transcriptase of usefulness American I nvitrogen also synthesizes cDNA. with reference to the product description reverse transcription
5) pcr amplification of mVEGF
CDNA is a template with step 4) reverse transcription synthetic, under the guiding of primers F 6 and R6, carry out pcr amplification, 100 μ l PCR reaction systems are: 2 * GC damping fluid I (TaKaRa company, La Taq is subsidiary) 50.0 μ l, dNTPs mixture (2.5mM) 16.0 μ l, TaKaRa La Taq (5U/ μ l) 1.0 μ l, reverse transcription product 2.0 μ l, F6 (20 μ M) 2.0 μ l, R6 (20 μ M) 2.0 μ l replenish cumulative volume to 100 μ l with the sterilization deionized water.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, detected result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 3; Swimming lane 1.PCR product), the result has obtained the gene fragment that length is about 288bp through pcr amplification, conforms to its result." 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 (available from TaKaRa company), check order to containing the pulsating recombinant vectors of recovery, extension increasing sequence is correct as a result.
The structure of embodiment 2, vascular endothelial growth factor VEGF-E antigen gene eukaryotic expression vector and evaluation thereof
One, the structure of vascular endothelial growth factor VEGF-E antigen gene eukaryotic expression vector
After the vascular endothelial growth factor VEGF-E antigen 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 '-Nhe I signal peptide-Xho I-EcoR V-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 * T4 DNA connects damping fluid 1 μ l, T4 dna ligase (12u/ μ l) 1 μ l, 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, get 1 μ l bacterium liquid and dilute, boil 10min, the centrifugal 3min of 12000rpm with 100 μ l water, get supernatant and do template, carry out pcr amplification under the guiding of primers F 5 and R5,25 μ l PCR 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 288bp through pcr amplification.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 the carrier for expression of eukaryon of vascular endothelial growth factor VEGF-E antigen gene, called after pCI-VEGFE.Also connect among the carrier pCI-GPI with the mVEGF gene fragment of same procedure simultaneously, obtain the mVEGF carrier for expression of eukaryon, called after pCI-mVEGFE the mouse of embodiment 1 amplification.
Two, the evaluation of eukaryon expression plasmid
1, transient transfection RenCa cell
The positive colony bacterium that carries carrier for expression of eukaryon pCI-VEGFE and pCI-mVEGFE respectively that step 1 is obtained is inoculated in the LB liquid nutrient medium, cultivated 12-24 hour down at 37 ℃, plasmid with QIAGEN company extracts test kit in a small amount and extracts plasmid with reference to product description, uses the transfection reagent of QIAGEN company then
Transfection Reagent and with reference to product description with pCI-VEGFE and pCI-mVEGFE plasmid transfection mouse renal carcinoma cell line RenCa, be 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-VEGFE plasmid to carry out RT-PCR with primers F 5 and R5 to transfection then identifies, there is the cell of pCI-mVEGFE plasmid to carry out RT-PCR with primers F 6 and R6 to transfection and identifies that qualification result is (swimming lane M.DNA relative molecular mass standard DL-2000 as shown in Figure 4; Swimming lane 1.pCI-VEGFE; Swimming lane 2.pCI-mVEGF), all obtained the fragment that length is about 288bp through amplification, show that the vascular endothelial growth factor VEGF-E antigen gene expresses on the mRNA level in transfection has the RenCa cell of pCI-VEGFE plasmid, the mVEGFE gene is also expressed on the mRNA level in transfection has the RenCa cell of pCI-mVEGFE 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 5; 2.pCI-mVEGF transfectional cell; 3.pCI-neo
+Transfectional cell; 4.RenCa cell), show that vascular endothelial growth factor VEGF-E antigen gene and mVEGF gene are obtaining to express in the cells transfected respectively separately.
3) the Western Blotting of stable transfected cells identifies
With step 2) stable transfected cells 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) use 0.25% trysinization, collecting cell, PBS washed twice; With the TBS that contains 1%Triton X-100 (50mM Tris-Cl, pH7.6,150mM NaCl) lysing cell 30min; 4 ℃, the centrifugal 10min collection of 12000rpm supernatant; After testing sample carried out 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 6; Swimming lane 2.pCI-mVEGF transfectional cell; Swimming lane 3.pCI-neo
+Transfectional cell; Swimming lane 4.RenCa cell), prove that further vascular endothelial growth factor VEGF-E antigen gene and mVEGF gene obtain respectively to express in transfectional cell separately.
One, the structure of vascular endothelial growth factor VEGF-E antigen gene prokaryotic carrier
Make up vascular endothelial growth factor VEGF-E antigen Prokaryotic Expression carrier referring to Fig. 7, concrete grammar is: the pCI-VEGFE plasmid 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 detect with 1.2% agarose gel electrophoresis, detected result is (swimming lane M1.DNA relative molecular mass standard DL-15000 as shown in Figure 8; Swimming lane M2.DNA relative molecular mass standard DL-2000; Before swimming lane 1.pGEX-4T-2 enzyme is cut; After swimming lane 2.pGEX-4T-2 enzyme is cut; Swimming lane 3.VEGF-E), 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 (the arrow indication fragment among Fig. 8) of the pCI-VEGFE of linearizing pGEX-4T-2 of purifying and 288bp, 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 288bp 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 primers F 5 and R5 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 vascular endothelial growth factor VEGF-E antigen Prokaryotic Expression carrier, called after pGEX-VEGFE.
Two, a large amount of preparations of the acquisition of engineering bacteria and inclusion body
With pGEX-VEGFE 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; Ultrasonic wave in the ice bath (30 seconds/open, 20 seconds/stop, 15 circulations) broken thalline; 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% (concentration expressed in percentage by volume) behind 20 ℃, the centrifugal 10min of 12000rpm; 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 of 32KD 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, detected result as shown in Figure 9, show and obtained the purified target protein of 32KD, i.e. vascular endothelial growth factor VEGF-E antigen.
The antitumor activity of embodiment 4, vascular endothelial growth factor VEGF-E antigen detects
One, grouping of animal and immunity
6 the week age Balb/c mouse be divided into 5 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 F-1 group is the pCI-mVEGF group; The F-2 group is the pCI-VEGFE group.
(pCI-neo, pCI-mVEGF (embodiment 2 makes up), pCI-VEGFE (embodiment 2 makes up)) presses 1: 1 (m: mixed v) with transfection reagent LipofectAMINE respectively with three kinds of plasmid DNA, 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 protein that embodiment 3 purifying are obtained 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 mouse isoantigen control group, this group mouse all produces specific antibody (P＜0.05)
※ ※Twice booster immunization be the antibody titer of enhancing immunity mice serum (P＜0.05) obviously
Compare with the empty carrier control group with PBS, all produced the antibody of higher level in the antigen group mice serum, twice booster immunization be the antibody titer of enhancing immunity mice serum obviously, in addition, the corresponding mouse isoantigen of vascular endothelial growth factor VEGF-E antigen 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 the RPMI1640 substratum that contains 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 10,
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
Compare with the empty carrier control group with PBS, the T lymphocyte quantity of secretion of gamma-IFN and IL-4 all obviously increases with the control group ratio in antigen group (F-1 group and the F-2 group) mouse boosting cell, and vascular endothelial growth factor VEGF-E antigen of the present invention more can excite the humoral immune reaction of mouse than the corresponding antigens 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/LNaN
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 11, shown in Figure 12.
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 (F-1 group and the F-2 group) mouse boosting cell
+/ CD8
+Ratio obviously raise, and vascular endothelial growth factor VEGF-E antigen of the present invention more can excite the humoral immune reaction of mouse than the corresponding antigens 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 and Figure 13, 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, antigen group (F-1 group and F-2 group) mouse become the knurl time lengthening, tumor growth is slow and knurl volume-diminished, tumour inhibiting rate obviously improve, and vascular endothelial growth factor VEGF-E antigen of the present invention is more remarkable than the tumor killing effect of the corresponding antigens of mouse.
Sequence table
<160>2
<210>1
<211>96
<212>PRT
＜213〉blue tongue virus
<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
<210>2
<211>288
<212>DNA
＜213〉blue tongue virus
<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?gcgattgt 288
Claims (10)
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| WO1999050290A1 (en) * | 1998-03-27 | 1999-10-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Para-poxvirus-coded vascular endothelial cell growth factor (ppv-vegf) |
| CN1259961A (en) * | 1997-04-07 | 2000-07-12 | 基因技术股份有限公司 | Humanized antibodies and method for forming same |
| WO2001062942A2 (en) * | 2000-02-25 | 2001-08-30 | Ludwig Institute For Cancer Research | MATERIALS AND METHODS INVOLVING HYBRID VASCULAR ENDOTHELIAL GROWTH FACTOR DNAs AND PROTEINS AND SCREENING METHODS FOR MODULATORS |
| WO2002081520A2 (en) * | 2001-04-06 | 2002-10-17 | Maxygen Holdings Ltd. | Single chain dimeric polypeptides derived from the vegf family |
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2006
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| CN1259961A (en) * | 1997-04-07 | 2000-07-12 | 基因技术股份有限公司 | Humanized antibodies and method for forming same |
| WO1999050290A1 (en) * | 1998-03-27 | 1999-10-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Para-poxvirus-coded vascular endothelial cell growth factor (ppv-vegf) |
| WO2001062942A2 (en) * | 2000-02-25 | 2001-08-30 | Ludwig Institute For Cancer Research | MATERIALS AND METHODS INVOLVING HYBRID VASCULAR ENDOTHELIAL GROWTH FACTOR DNAs AND PROTEINS AND SCREENING METHODS FOR MODULATORS |
| WO2002081520A2 (en) * | 2001-04-06 | 2002-10-17 | Maxygen Holdings Ltd. | Single chain dimeric polypeptides derived from the vegf family |
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