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Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor S2 immune decision cluster

A technology of hepatitis B surface antigen and fusion protein, which can be used in peptide/protein components, fusion polypeptides, genetic engineering, etc., and can solve problems such as easy degradation and peptide bond breakage.

Inactive Publication Date: 2008-08-27
CHENGDU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the natural preS2 region contains multiple protease-sensitive sites, especially the peptide bond between the 48th arginine and the 49th threonine at the N-terminus is easily broken by proteases (Langley KE, Egan KM, Barendt JM , et al Characterization of purified hepatitis B surfaceantigen containing pre-S(2) epitopes expressed in saccharomyces cerevisiae.Gene1988, 67 229-245), causing the preS2 antigen to be easily degraded and inactivated during the process of expression and preparation

Method used

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  • Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor S2 immune decision cluster
  • Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor S2 immune decision cluster
  • Hepatitis B surface antigen blending gene and protein for carboxyl terminal containing precursor S2 immune decision cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] [Example 1] Synthesis of the hepatitis B surface antigen fusion gene containing the pre-S2 epitope at the carboxyl end and construction of the expression vector

[0023] 1. Main materials and reagents

[0024] Pichia pastoris strain GS115 (His - ,Mut + ), Escherichia coli Top10F' and Pichia pastoris expression plasmid pAO815 were purchased from Invitrogen Company, and Escherichia coli DH5α and cloning vector pBluescript II SK (+) were common bacterial strains and plasmids. Restriction enzymes and Taq DNA polymerase were purchased from NEB Company, calf intestinal alkaline phosphatase (CIP), T4 DNA ligase was purchased from MBI Company, and DNA fragment recovery kit was purchased from Boehringer Mannheim Company

[0025] 2. Synthesis of PCR primers and amplification of SS2 fusion gene fragment

[0026] Hepatitis B virus (adr subtype) gene sequence, synthesize the following four primers:

[0027] P1: 5'-AAAGAATTCACCATGGAGAACACAGCATCA-3';

[0028] P2: 5'-AATGTATACCCA...

Embodiment 2

[0054] [Example 2] Expression of the hepatitis B surface fusion antigen SS2 containing the pre-S2 antigenic determinant at the carboxyl end in Pichia Pastoris (Pichia Pastoris)

[0055] 1. Main materials and reagents

[0056] Hepatitis B surface antigen reversed-phase hemagglutination (RPHA) kit and surface antigen (S) enzyme-labeled kit were purchased from Shanghai Kehua Biotechnology Co., Ltd., hepatitis B surface antigen S and pre-S2 antibodies were purchased from Oxford Biotechnology Limited and Orbigen Inc. product.

[0057] 2. Transformation of recombinant plasmids into yeast cells and preliminary screening of positive clones

[0058] The pAO815-SS2 plasmid was linearized with Bgl II, extracted with phenol / chloroform and precipitated with alcohol, and then transformed into GS115 cells by electroporation. With MD [1.34% YNB (without amino acid), 4×10 -5 % biotin, 2% glucose] agar plate to screen positive clones. Since the GS115 strain itself cannot synthesize histidin...

Embodiment 3

[0068] [Example 3] engineering bacteria fermentation and compound antigen purification

[0069] 1. Main materials and reagents

[0070] MGY medium: 1.34% yeast nitrogenous basic medium, 1% glycerol, 4*10 -5 % Biotin;

[0071] Basic salt solution:

[0072] Phosphoric acid 85% 26.7ml

[0073] Calcium sulfate 0.93g

[0074] Potassium sulfate 18.2g

[0075] Magnesium sulfate 7 water 14.9g

[0076] Potassium hydroxide 4.13g

[0077] Glycerin 40.0g

[0078] Distilled water to make up to 1L;

[0079] PTMs 1 Trace salt solution:

[0080] Copper sulfate 5 water 6.0g

[0081] Sodium iodide 0.08g

[0082] Manganese sulfate 1 water 3.0g

[0083] Sodium molybdate dihydrate 0.2g

[0084] Boric acid 0.02g

[0085] Cobalt chloride 0.5g

[0086] Zinc chloride 20.0g

[0087] Ferrous sulfate 7 water 65.0g

[0088] Biotin 0.2g

[0089] Sulfuric acid 5.0ml

[0090] Distilled water to make up to 1L;

[0091] 2. Engineering bacteria fermentation

[0092] Pick a single colony of...

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Abstract

The invention provides a fusion gene encoding a new-type Hepatitis B virus surface antigen and the protein thereof. The nucleotide sequence, encoding Hepatitis B virus preS2 antigen (Met1-Gly26) containing B-cell and T-cell epitopes and polymerized albumin binding site, is fused to 3' terminal of Hepatitis B virus surface antigenic main protein S gene, then the expression is carried out in yeast expression system. The expressed products, possessing simultaneously antigenicity of S and preS2, are assembled into virus-like particles with diameter of about 20-35nm and are potential for developing novel Hepatitis B vaccines.

Description

technical field [0001] The invention relates to a new hepatitis B surface antigen coding gene, in particular to a hepatitis B surface antigen fusion gene constructed by a genetic engineering method containing a pre-S2 immune determinant at the carboxyl end. [0002] The invention also relates to the recombinant vector of the gene and the encoded protein. Background technique [0003] Hepatitis B is a serious infectious disease caused by the hepatitis B virus, which is widely distributed around the world. It is estimated that there are about 120 million hepatitis B virus carriers in China alone, of whom about 10 million are patients . Currently there is no effective treatment for hepatitis B, and hepatitis B vaccination is an effective way to prevent and control hepatitis B. [0004] Early hepatitis B vaccines were made from HBsAg particles isolated from the plasma of hepatitis B virus carriers. Due to limited plasma sources, coupled with cost and safety considerations, thi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/63C12N15/51C07K14/02A61K38/16A61K39/29A61P1/16
CPCC07K2319/00A61K2039/5258C12N2730/10122C07K14/005C12N2730/10134A61K39/292A61K39/12A61P1/16
Inventor 谭昌耀蒋丽明葛永红袁进金瓯
Owner CHENGDU INST OF BIOLOGICAL PROD
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