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Recombinant protein vaccine, recombinant expression vector containing genes for coding recombinant protein vaccine and application of recombinant protein vaccine

A technology of recombinant protein and expression vector, applied in the biological field, can solve the problems of single EB virus protein, eliminate EB virus, etc., and achieve the effect of preventing EB virus infection, eliminating diseases and tumors, and preventing or treating EB virus infection

Active Publication Date: 2015-06-17
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 genes for coding recombinant protein vaccine and application of recombinant protein vaccine
  • Recombinant protein vaccine, recombinant expression vector containing genes for coding recombinant protein vaccine and application of recombinant protein vaccine
  • Recombinant protein vaccine, recombinant expression vector containing genes for coding recombinant protein vaccine and application of recombinant protein vaccine

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Embodiment 1

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

[0060] (1) Cloning of Epstein-Barr virus nuclear antigen 1 gene sequence

[0061] Using the extracted EBV genome as a template, primers were used: upstream primer EBNA1-F:5'-ATGTCTGACGAGGGGCCAGG-3' (SEQ ID No7) and downstream primer EBAN1-R:5'-CTCCTGCCCTTCCTCACCCT-3' (SEQ ID No.8) EBNA1 was amplified by PCR, and the amplification conditions were:

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

[0063] The reaction parameters were: 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 8min.

[0064] Then, the PCR product was recovered by agarose gel electrophoresis, and the recovered product was sent to Yingwei Jieji (Shanghai...

Embodiment 2

[0096] This embodiment provides a method for expressing and purifying a recombinant protein vaccine, comprising the following steps:

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

[0098] Transform the pET28a-gD-EBNA1 plasmid into BL21 expression bacteria according to the conventional molecular cloning method in the laboratory to obtain the E.Coli-pET28a-gDEBNA1 strain;

[0099] In 5ml of LB liquid medium containing 50mg / L Kan, inoculate the E.Coli-pET28a-gDEBNA1 bacterial solution at a ratio of 1:50, and culture overnight at 37°C with shaking at 220rpm; the next day, inoculate the cultured bacterial solution overnight until it contains 50mg / L In Kan's new LB liquid medium, culture at 37°C with shaking at 220rpm 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; after induction for 2, 4, and 6 hours, respectively Finally, collect the bacterial liquid, collect the bacterial cells by high-speed centr...

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Abstract

The invention provides a recombinant protein vaccine. The recombinant protein vaccine contains an epitope of EB virus antigen 1 (EBNA1) and an epitope of human herpes virus glycoprotein; fusion proteins can effectively activate a specific cell toxic T lymphocyte (CTL) reaction, directly restrain the expression EBNA1, and give play to treatment effects on EB virus-associated tumors; an immunosuppression path can be directly blocked through herpes virus glycoprotein, and the activity of regulatory T cells is improved. In this way, by means of the recombinant protein vaccine, EB virus infections and diseases related to the EB virus infections and tumors related to the EB virus infections can be prevented or treated in a multi-way manner more comprehensively. The invention further provides a recombinant expression vector containing genes for coding the recombinant protein vaccine and 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 γ-herpes virus with a 172kb DNA genome, which is classified as the first type of tumorigenic virus by the World Health Organization. People who have been infected with Epstein-Barr virus before the age of 10, but do not cause symptoms, can establish latent infection in B lymphocytes and stimulate cell proliferation and transformation. Epstein-Barr virus is related to the occurrence of various diseases, such as nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's lymphoma, gastric cancer, and acquired immunodeficiency syndrome-related central nervous system lymphoma. [0003] According to the current research progress, Epstein-Barr virus nuclear antigen 1 (EBNA1) is a DNA-binding protein encoded by t...

Claims

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Application Information

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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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