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Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof

A technology of forest encephalitis virus and fusion protein, applied in the preparation of forest encephalitis vaccine, the field of fusion protein of forest encephalitis virus envelope E protein and human antibody IgG G1 Fc segment, can solve the problem that there is no recombinant vaccine report, etc. problem, to achieve the effect of improving antibody and cellular immune response, enhancing immunogenicity, and increasing expression level

Inactive Publication Date: 2011-10-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report of a recombinant vaccine based on the envelope E protein as the target antigen

Method used

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  • Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof
  • Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof
  • Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1. Synthesis of the envelope E gene of forest Zhang strain forest encephalitis virus in my country:

[0041] According to the amino acid sequence (GenbankAccession: AY182009) encoded by the E gene of my country's Senzhang strain forest encephalitis virus, an optimized E gene sequence (as shown in SEQ ID NO: 1) was designed by using the codons preferred by mammalian cells, of which 5 The 'end contains the NheI restriction site GCTAGC and the initiation codon ATG, and the 3' end contains the BamHI restriction site GGATCC. Shanghai Jierui Biotechnology Co., Ltd. was entrusted to synthesize the gene by conventional overlap extension polymerase chain reaction. Insert the cloning vector T vector (product of Promega, USA) and name it pGEM-E.

Embodiment 2

[0042] Example 2. Cloning of human antibody IgG1 Fc segment gene:

[0043] B cells were isolated from 20 ml of fresh human plasma with magnetic beads labeled with CD19 monoclonal antibody (product of Miltenyibiotec, Germany), and the operation was performed according to the instructions for use. The total cellular RNA was extracted with Trizol Reagent (product of Invitrogen, USA), and the operation was performed according to the instructions for use. The obtained total cellular RNA was dissolved in DEPC-treated sterile double-distilled water. Then reverse transcription reaction (RT)-polymerase chain reaction (PCR) was used to amplify the human IgG1 Fc segment gene. The reagents used are products of Promega Company, USA. Take 2 μl of RNA (about 5 μg), put it in a 0.5 ml centrifuge tube, and then add the following components: 5 μl AMV reverse transcription reaction buffer, 1 μl dNTP (concentration 10 mM), 1 μl oligo(dT)15 (concentration 5 pmol / μl), Place in a hot water bath a...

Embodiment 3

[0044] Example 3. Construction of fusion gene expression plasmid of forest encephalitis virus envelope E and human IgG1 Fc segment:

[0045] Plasmid pGEM-E was digested with NheI and BamHI, and the E gene fragment of 1400 base pairs was recovered by agarose gel electrophoresis. The plasmid pGEM-Fc was digested with BamHI and SalI, and the Fc gene fragment of 700 base pairs was recovered by agarose gel electrophoresis. The CHO cell expression vector pCIDA-GS-neo constructed by the inventor with independent intellectual property rights was digested with NheI and SalI (see the Chinese patent ZL 200610024202.5 authorized by the applicant for the construction method, and the name of the invention is: a mammalian The method for efficiently secreting and expressing the envelope protein E2 of hepatitis C virus by cells, the inventors are: Zhao Ping, Liao Xiaoling, Cao Jie, Qi Zhongtian.), agarose gel electrophoresis to recover the linearized vector, and the recovered linearized vector...

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Abstract

The invention relates to the technical field of biomedical engineering, a tick-borne encephalitis virus is a neurotropic virus with extremely strong toxicity, and the tick-borne encephalitis caused by the tick-borne encephalitis virus is an acute infectious disease of a central nervous system. At present, both the crude vaccines and purified vaccines for the tick-borne encephalitis are inactivated vaccines, and have the problems of complicated antigen components, low content of effective antigen, multiple side actions and the like. Envelop E protein is an important structural protein of the tick-borne encephalitis virus, and a neutralizing antibody induced by the envelop E protein is a main immunoprotection mechanism of an organism against the tick-borne encephalitis virus. The invention utilizes a molecular cloning method to construct a fusion protein of the tick-borne encephalitis virus envelop E protein and human IgG1 (immune gamma globulins 1) Fc fragment, and then carries out transfection on CHO (Chinese hamster ovary) cells, thus expressing a recombinant fusion protein. The invention proves that the fusion with human IgG1 Fc fragment can obviously improve the expression level of the E protein and facilitate the purification of protein, and can obviously improve the immunogenicity of the E protein and enhance the immune response of E antibody and cells induced by the E protein. The fusion protein provided by the invention has a good application prospect in being used as an antigen for the tick-borne encephalitis vaccines.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a fusion protein of forest encephalitis virus envelope E protein and human antibody IgG G1 Fc segment, and its application in the preparation of forest encephalitis vaccine. Background technique [0002] Forest encephalitis virus, also known as tick-borne encephalitis virus (TBEV), is a highly virulent neurotropic virus that mainly invades the central nervous system of humans, and forest encephalitis caused by it is an acute central nervous system infection. Systemic infectious diseases, fatality rate and disability rate are quite high. Forest encephalitis virus is one of the important viral biological warfare agents, and the US National Center for Disease Control classifies the virus as a category C virus biological weapon. In my country, forest encephalitis is a legal occupational disease caused by biological factors. Forest encephalitis vaccine is one of the n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/12A61P31/14
CPCY02A50/30
Inventor 赵平朱诗应任浩戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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