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Recombinant human VEGF tumor polypeptide vaccine

A tumor vaccine, tumor technology, applied in the field of medical biotechnology and immunology, can solve the problem of less immunogenicity

Pending Publication Date: 2017-06-09
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the drawback of peptide vaccines is that unmodified peptides are rarely immunogenic

Method used

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  • Recombinant human VEGF tumor polypeptide vaccine
  • Recombinant human VEGF tumor polypeptide vaccine
  • Recombinant human VEGF tumor polypeptide vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Construction of recombinant human VEGF polypeptide vaccine prokaryotic expression plasmid

[0015] A synthetic method was used to obtain the C-terminal with Bam H I and EcoR The phage Qβ coat protein gene expression sequence at the Ⅰ restriction site was cloned into the first expression unit of the vector pRSFDuet1, and the synthetic upstream was introduced Bam HI, downstream introduction Eco The human VEGF antigen peptide nucleic acid sequence of the RI restriction site is connected to the C-terminus of the Qβ coat protein gene expression sequence; the phage Qβ coat protein gene expression sequence is inserted into the second expression unit of the vector pRSFDuet1.

Embodiment 2

[0016] Example 2: Prokaryotic expression of recombinant human VEGF polypeptide vaccine

[0017] Transform the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells, pick a single clone into LB medium containing kanamycin (50mg / mL), culture overnight for 12-16 hours, and then press the ratio of 1:20 Transfer to fresh LB medium containing kanamycin and continue culturing for 2-3 hours. When the OD value of the bacterial solution reaches 0.5, add IPTG (1 mmol / L) to induce expression for 3 hours, and the induction temperature is 37°C.

Embodiment 3

[0018] Embodiment 3: Purification of recombinant human VEGF polypeptide vaccine

[0019] The induced bacterial solution was centrifuged at 4500 rpm for 20 min to collect the bacterial cells, and the bacterial cells were resuspended in PBS and then ultrasonically disrupted under the conditions of 15% power, working for 5 sec, stopping for 5 sec, and the total time was 10 min. The ultrasonically crushed sample was centrifuged at 12000 rpm for 10 min, the supernatant was added with ammonium sulfate to 40% saturation, mixed for 30 min at 4 °C, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, the precipitate was dissolved in PBS, and the lysate was dissolved in 10 min -50% sucrose solution was subjected to density gradient centrifugation (40,000 rpm, 3 h), and the bands containing virus-like particles were taken out, and the sucrose was replaced with PBS through a G-25 desalting purification column to obtain a purified recombinant human VEGF polypeptide vaccine ( ...

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Abstract

The invention discloses a recombinant human VEGF tumor polypeptide vaccine. Human VEGF epitope nucleotide sequence is acquired by a synthesizing process and is inserted to the terminal C of coat protein gene of bacteriophage QBeta. Through a coexpression system, coat protein gene of bacteriophage QBeta and the coat protein gene of bacteriophage QBeta of which the terminal C is inserted with the VEGF epitope are expressed simultaneously in escherichia coli BL21(DE3), and QBeta virus-like particles with human VEGF epitope presented on surfaces are obtained after the expression product is purified. The recombinant human VEGF-QBeta protein produces human VEGF antibody through immunized mice; the human VEGF antibody is capable of neutralizing biological activity of human VEGF 165 when applied to the HUVEC cell proliferation test. When applied to immunization of mice, the homologous mouse VEGF polypeptide vaccine which is produced by the presenting way has a certain inhibition effect to TC-1 tumor cells in bodies of C57 BLACK6 mice.

Description

technical field [0001] The invention relates to the fields of medical biotechnology and immunology, in particular to a construction method of a human VEGF polypeptide vaccine. [0002] technical background [0003] Studies have shown that angiogenesis plays an important role in the occurrence and development of tumors, and inhibiting tumor angiogenesis has become a new treatment method. Many active factors have been confirmed to be involved in this complex process, including: aFGF, bFGF, TGF-α, VEGF and so on. However, research done over the past few years has established that in tumor angiogenesis, vascular endothelial growth factor (VEGF) is one of the most potent and specific pro-angiogenic factors known. In most tumors, VEGFmRNA is overexpressed and thus considered to be the most effective target for inhibiting tumor angiogenesis. [0004] Various strategies have been used to inhibit the effects of VEGF, including targeting the VEGF receptor (VEGFR), using gene therapy ...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/39A61P35/00
CPCA61K39/0011A61K39/39A61K2039/55505
Inventor 马雁冰白红妹黄惟巍夏烨刘存宝孙文佳杨旭
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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