Hepatitis E virus-like particle vaccine preparation method and application

A technology of hepatitis E virus and particles, which is applied in the field of biomedicine and can solve problems such as unclear functions and complex manufacturing processes of virus-like particles

Active Publication Date: 2015-08-26
张澍 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Attenuated or inactivated vaccines have not been developed because there is no suitable cell system. Only China has developed and registered the HEV virus-like particle vaccine expressed by Escherichia coli. However, the expressed virus-like particle has a complicated manufacturing process and the expressed protein is inclusion body. Complex denaturation process required for assembly into virus-like particles
Among the HEV proteins, ORF1 encodes a non-structural protein, which does not form neutralizing antibodies during immunization, and ORF3 encodes a non-structural protein with unclear functions, which forms antibodies for a short period of time, but has no neutralizing effect

Method used

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  • Hepatitis E virus-like particle vaccine preparation method and application
  • Hepatitis E virus-like particle vaccine preparation method and application
  • Hepatitis E virus-like particle vaccine preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Hepatitis E virus capsid protein gene codon optimization

[0067] Reagents and enzymes used: T4 DNA ligase and restriction endonuclease were purchased from NEB Company in the United States, Isopropyl-β-D-thiogalactopyranoside (IPTG), polymerase, dNTP and protein markers were purchased from Roche Company in Germany, and the bioreactor was purchased from Lithuania Company, all chemicals were purchased from German Merck (Germany) and American Sigma Company.

[0068] The genome sequence of HEV gene ORF2 was obtained from the GenBank China HEV isolate (accession no.AF444002.1). The wild-type truncated ORF2.1 encodes the 1-660 amino acids of the HEV capsid protein. GenScript software was used to analyze the rare codons of the wild-type ORF2 genome sequence, and two restriction enzymes NdeI and XbaI were placed at both ends of the ORF2 gene . The pGEM-T plasmid was digested according to the restriction sites shown in Table 1 to synthesize the full-length ORF2 gene ...

Embodiment 2

[0074] Soluble, massive expression and extraction of the capsid protein HEV-ΔORF2 protein of embodiment 2 hepatitis E virus truncated

[0075] 1 Test method

[0076] 1.1 Construction and screening of soluble expression plasmids

[0077] The ΔORF2 gene product synthesized in Example 1 was ligated with the pMAL-p4x expression plasmid fragment digested with the same restriction enzymes. In order to increase the solubility of the expression product, select a suitable fusion partner and purification tag, especially the optimized fusion partner is the N-terminal maltose-binding protein, and no fusion partner or purification tag is added to the C-terminus to ensure that the recombinant expression product can be used as a vaccine antigen. The MBP (maltose binding protein) tag protein is 40kDa in size and is encoded by the malE gene on the pMAL-p4x expression plasmid. MBP can increase the solubility of fusion proteins overexpressed in bacteria, especially eukaryotic proteins. The MB...

Embodiment 3

[0099] Example 3 Hepatitis E virus truncated capsid protein HEV-ΔORF2 proteolytic digestion and affinity chromatography purification

[0100] 1 Test method

[0101] 1.1 Digestion of MBP-ΔORF2 fusion protein

[0102] Factor Xa was prepared in buffer (20mM Tris, pH 8.0, 0.2M NaCl, 5mM CaCl 2 ), adding 1 unit of factor Xa to 100 μg of protein, adding factor Xa to the HEV-ΔORF2 protein prepared in Example 2, acting at room temperature for 36-48 hours, and digesting 95% of the protease.

[0103] 1.2 Purification of anti-maltose antibody on Sepharose 4 affinity medium

[0104] Enzyme digestion reaction solution and 5mL anti-maltose antibody-coupled Sepharose 4 affinity medium were mixed and incubated at 4°C for 18-24 hours, and 0.5mg MBP was adsorbed per 1mL medium, which could be covalently bound to specific sites on MBP for purification and MBP fusion expressed protein. The supernatant was harvested after centrifugation at 4000 rpm at room temperature.

[0105] The protein in...

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Abstract

The invention discloses a hepatitis E virus-like particle vaccine preparation method, and belongs to the biological medicine technical field. According to the method, hepatitis E virus truncated capsid protein gene codon optimization is performed, prokaryotic expression system escherichia coli strain B834-pRARE2 is used, by fusion of purification tag MBP expression, different cracking agent screening and combination, bacterial membrane extraction, maltose amylose affinity chromatography, molecular sieve chromatography for removal of MBP and non-virus structural protein, and promotion of virus-like particle assembly, the hepatitis E virus-like particle purity is more than 99.0%, and is free of protein label and bioactive (in the form of non-inclusion body, and soluble). The hepatitis E virus-like particle vaccine produces complete protection to pig, dog and chicken HEV infection, cross protective antigens are between different genotype HEV virus, and the hepatitis E virus-like particle vaccine can be used as an animal vaccine to prevent the diseases and infection caused by pig, dog and poultry HEV.

Description

technical field [0001] The invention relates to a method for preparing a hydrophobic virus capsid protein and its application, in particular to a method for preparing a hepatitis E virus-like particle vaccine and its application, and belongs to the technical field of biomedicine. Background technique [0002] Hepatitis E virus (HEV) is a member of the Hepavirus genus in the family Hepadnaviridae. It contains a forward-sense single-stranded RNA and has three overlapping open reading frames (ORFs) in its genome, including ORF1, ORF2, and ORF3. Viruses are divided into four genotypes, genotype I and genotype II viruses are mainly isolated from humans, genotype III and genotype IV viruses are mainly derived from pigs, chickens, rabbits, dogs and other animals, and even genotype V viruses have been found. Genotype I viruses are mainly prevalent in China and Southeast Asia. HEV is the main pathogen of acute hepatitis, causing moderate and severe hepatitis, and the severity increas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/29A61K39/42C07K16/10A61P31/14A61P1/16
Inventor 吕宏亮张澍
Owner 张澍
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