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Recombinant protein vaccine, recombinant expression vector containing gene encoding the recombinant protein vaccine and application thereof

A recombinant protein and expression vector technology, applied in the biological field, can solve problems such as eliminating EB virus and single EB virus protein, and achieve the effects of preventing EB virus infection, preventing or treating EB virus infection, and improving activity

Active Publication Date: 2017-11-07
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, EBV-specific antigen peptide vaccines are a hot spot in the field of tumor vaccine research. They mainly target specific targets in EB virus infection or incubation period, such as gp350, LMP1, LMP2 and EBNA3A, etc., and develop vaccines targeting these targets. Through Inhibit the expression of the corresponding protein to achieve the purpose of treating Epstein-Barr virus-related tumors. However, the vaccines in the existing research only target a single Epstein-Barr virus protein, and most of the target antigens are only expressed in a specific period of virus infection, which cannot be comprehensive and effective. Effectively resist Epstein-Barr virus infection, eliminate Epstein-Barr virus and its related diseases and tumors

Method used

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  • Recombinant protein vaccine, recombinant expression vector containing gene encoding the recombinant protein vaccine and application thereof
  • Recombinant protein vaccine, recombinant expression vector containing gene encoding the recombinant protein vaccine and application thereof
  • Recombinant protein vaccine, recombinant expression vector containing gene encoding the recombinant protein vaccine and application thereof

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Embodiment 1

[0059] A method for constructing a recombinant expression vector containing a nucleotide sequence encoding the recombinant protein vaccine of claims 1 to 5, comprising the following steps:

[0060] (1) Cloning of EB virus nuclear antigen 1 gene sequence

[0061] Using the extracted EBV genome as a template, primers: upstream primer EBNA1-F: 5'-ATGTCTGACGAGGGGCCAGG-3' (SEQ ID No 7) and downstream primer EBAN1-R: 5'-CTCCTGCCCTTCCTCACCCT-3' (SEQ ID No. 8) PCR amplification of EBNA1, the amplification conditions are:

[0062] PCR reaction system (100μl): 5╳Buffer 20μl, dNTP mix 8μl, DNA 6μl, Taq enzyme 1μl, primer mixture 4μl, double distilled water 61μl;

[0063] The reaction parameters are: 95°C for 2min, 95°C for 15s, 68°C for 15s, 72°C for 3min, a total of 35 cycles, and finally 72°C for 8 minutes.

[0064] Then, the PCR product was recovered by agarose gel electrophoresis, and the recovered product was sent to Invitech (Shanghai) Trading Co., Ltd. for sequencing. The sequence obtained...

Embodiment 2

[0096] This embodiment provides a method for expression and purification of a recombinant protein vaccine, including the following steps:

[0097] (1) Expression of His-Tag tag in E. coli

[0098] Transform the pET28a-gD-EBNA1 plasmid into BL21 expressing bacteria according to the routine methods of laboratory molecular cloning to obtain E.Coli-pET28a-gDEBNA1 strain;

[0099] In 5ml of LB liquid medium containing 50mg / L Kan, inoculate E.Coli-pET28a-gDEBNA1 bacterial solution at a ratio of 1:50, cultivate overnight at 37°C and 220rpm; inoculate the overnight bacterial solution the next day to contain 50mg / L In Kan's new LB liquid medium, culture at 37°C and 220rpm with shaking to OD600=0.6~0.8; add IPTG to each tube to a final concentration of 0.5mmol / L, and induce expression at 28°C; induce expression at 2, 4, and 6 hours respectively Then collect the bacteria liquid, collect the bacteria by high-speed centrifugation at 12000g at 4℃; add 2ml phosphate buffer to resuspend and wash th...

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Abstract

The invention provides a recombinant protein vaccine, which contains the epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1) and the epitope of human herpes virus glycoprotein, and the fusion protein can not only effectively activate specific cytotoxicity T lymphocyte (CTL) reaction, directly inhibits the expression of EBNA1, exerts the therapeutic effect of Epstein-Barr virus-related tumors; it can also directly block the immunosuppressive pathway through the herpes virus glycoprotein, and improve the activity of regulatory T cells; therefore, the present invention provides The recombinant protein vaccine can prevent or treat Epstein-Barr virus infection and related diseases and tumors more comprehensively and in multiple ways; the invention also provides a recombinant expression vector containing the gene encoding the recombinant protein vaccine and the application of the recombinant protein vaccine.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant expression vector containing a gene encoding the recombinant protein vaccine and its application. Background technique [0002] Epstein-Barr Virus (EBV) is a human gamma herpes virus containing a 172kb DNA genome. It is classified as the first type of oncogenic virus by the World Health Organization. The infection is very common in the population. More than 90% of individuals He has been infected with Epstein-Barr virus before the age of but does not cause symptoms. It can establish latent infection in B lymphocytes and stimulate cell proliferation and transformation. Epstein-Barr virus is related to the occurrence of many diseases, such as nasopharyngeal cancer, infectious mononucleosis, Hodgkin's lymphoma, gastric cancer, acquired immune deficiency syndrome-related central nervous system lymphoma, etc. [0003] Up to the present research progress, Epstein-Barr virus nuclear...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/295A61K39/245A61P31/22C12N15/66C12N15/70C12N1/21C12R1/19
Inventor 王蒲邹军辉唐超万晓春
Owner SHENZHEN INST OF ADVANCED TECH
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