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Recombinant protein and expressing method thereof in insect baculovirus expression system

A technology for recombining baculoviruses and proteins, applied in the field of genetic engineering, can solve problems such as unclear biological functions

Inactive Publication Date: 2015-08-12
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HBV pre-c protein (hepatitis B virus precore, HBV pre-c) is the precursor of hepatitis B virus c protein and e protein, and its biological function is not yet clear

Method used

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  • Recombinant protein and expressing method thereof in insect baculovirus expression system
  • Recombinant protein and expressing method thereof in insect baculovirus expression system
  • Recombinant protein and expressing method thereof in insect baculovirus expression system

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preparation example Construction

[0044] The preparation of recombinant virus can be prepared by various consistent methods in the art. The present invention optimizes the Bac-Bac system, utilizes the carrier Bacmid that can shuttle between Escherichia coli and insect cells, and transforms the sequence of HBV pre-c-Fc protein into baculovirus, thereby being able to highly express HBV pre-c-Fc in insect cells. c-Fc protein.

[0045] In accordance with the present invention, conventional molecular biology, microbiology, cytology and DNA recombinant techniques within the skill of the art may be employed. Definitions of terms where they appear in this document are as follows.

[0046] "DNA molecule" refers to the polymeric form of deoxyribonucleotides (thymine, cytosine, adenine or guanine), which are the main constituents of chromosomes and the material that makes up genes. This term refers only to the primary and secondary structure of the molecule, without restricting it to any specific tertiary form. The te...

Embodiment 1

[0058] 1. the gene sequence of the hepatitis B virus pre-C protein of the present invention and the gene sequence of the mouse IgG Fc protein all come from NCBI ( http: / / www.ncbi.nlm.nih.gov / ), the gene sequence of the hepatitis B virus pre-C protein and the gene sequence of the mouse IgG Fc protein are directly combined, but the IgG Fc gene sequence of the mouse also includes the connexin sequence in front of it, and the sequence is not changed as a whole, and the obtained Polynucleotide sequence as shown in SEQ ID NO:1, then codon optimization is carried out to SEQ ID NO:1, obtain the polynucleotide sequence as shown in SEQ ID NO:2, codon website http: / / www. kazusa.or.jp / codon / cgi-bin / showcodon.cgi? species=10455: to facilitate the expression of this sequence in insect cells. The codon optimization of the present invention is directly optimized by Huada Gene Company, and then fully synthesized. The specific optimized codon table is as follows

[0059] Spodoptera frugipe...

Embodiment 2

[0109] Example 2 Expression and identification of HBV pre-c-Fc protein

[0110] 1. Expression and identification of HBV pre-c-Fc protein Suspension culture insect cells in logarithmic growth phase, cell density 2×10 6 pieces / ml. Transfection with P4 virus, 27°C, no CO 2 , 110rpm / min suspension culture. Cell supernatants were harvested at 36, 48, 60, 72, 84, 96, 108, and 120 hours, respectively. Five flasks of cells in the same growth state were taken, and the cells were infected with different MOIs, and the protein was harvested after 84 hours. Exposure was detected by Western Blot.

[0111] The composition of the protein loading buffer is: 5× protein loading buffer, the configuration method is: measure 1M Tris-HCl (pH6.8) 0.6ml; 50% glycerin 5ml; 10% SDS solution 2ml; 1% bromophenol blue 1ml; add Deionized water Dilute to 10ml; dispense 200μl per tube; store at 4°C, add 50μl of β-mercaptoethanol before use.

[0112] When used with samples, use protein loading buffe...

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Abstract

The invention discloses HBV pre-c-Fc recombinant protein and a coding nucleotide sequence thereof and discloses a recombinant vector containing a coding sequence of the protein and a host cell. The invention further discloses a recombinant baculovirus, a method of obtaining the recombinant baculovirus, and a method of expressing the HBV pre-c-Fc recombinant protein in an insect baculovirus expression system, and also discloses a recombinant baculovirus containing a HBV pre-c-Fc coding gene and applications of the HBV pre-c-Fc recombinant protein in preparation of vaccines or medicines preventing or treating hepatitis B. After a BALB / c mouse is subjected to injection immunization with the HBV pre-c-Fc recombinant protein, detection shows that the generating amount of a hepatitis B virus core protein antibody in serum of the mouse is far higher than that in a situation that an antigen used for preparing hepatitis B vaccines in traditional methods is used.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant protein and its expression method in an insect baculovirus expression system. Background technique [0002] Hepatitis B virus (HBV) infection is one of the global disease burdens, especially in my country: more than 200 million people around the world have been or are being infected with HBV, which can cause hepatitis, cirrhosis and even hepatocellular carcinoma ( Hepatocellular carcinoma, HCC). According to statistics, about 780,000 people die from hepatitis B and its related diseases every year, seriously endangering human health and affecting people's quality of life. The entire HBV genome can encode a variety of viral proteins, and in-depth exploration of the biological functions of various viral proteins is of great significance for revealing the pathogenesis of HBV infection-related diseases and guiding clinical detection and treatment. [0003] At present,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/866A61K39/29A61K47/48A61P31/20
Inventor 姜潮李校堃马吉胜田海山刘敏王海军王晓艳宋娜
Owner WENZHOU MEDICAL UNIV
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