Hepatitis E virus ORF2 protein

A technology of ORF2 and hepatitis E virus, which is applied in the field of vaccine protein screening, can solve problems such as interference with epidemic monitoring, difficulty in cultivating virus strains, and affecting the protection effect of virus attack, and achieves convenient and accurate detection methods, high antigen stability, and specificity strong effect

Inactive Publication Date: 2019-11-22
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional inactivated vaccines have unique advantages such as uniformity of antibodies after immunization and high antibody titers, but there are also difficulties in cultivating the strains used to make vaccines, and there is a large difference in antigenicity from clinically popular strains, which affects the challenge. The shortcomings of the protective effect restrict the application of inactivated vaccines
Moreover, antibodies produced after immunization with traditional vaccines prepared using whole virus antigens cannot be clinically distinguished from wild virus infection or vaccine immunization, which will interfere with the monitoring of the epidemic

Method used

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  • Hepatitis E virus ORF2 protein
  • Hepatitis E virus ORF2 protein
  • Hepatitis E virus ORF2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Amplification and sequence analysis of ORF2 gene

[0015] In 2018, suspected hepatitis E occurred in some broiler breeders from Dalian area. After clinical investigation and laboratory testing, it was initially diagnosed as hepatitis E. After investigation, the above-mentioned infected chickens had been injected with hepatitis E vaccine and tested for neutralizing antibodies. It was suspected that the hepatitis E virus had mutated under the selection pressure of the vaccine, and the hepatitis E virus QD07 strain was isolated from the diseased chicken sample as a template for amplification.

[0016] 1. Amplification of the HEV ORF2 gene

[0017] According to the hepatitis E virus ORF2 gene sequence published in NCBI, primers were designed and synthesized. The sequence information of the primers is as follows:

[0018] primer1: 5′-ATGTCGGTGCGTGGATTGTTGCTC-3′;

[0019] primer2: 5'-CTAGGGTGGTGAGGGAAACGT-3'.

[0020] Extract the nucleic acid of the isolated...

Embodiment 2

[0027] Example 2: Recombinant expression of hepatitis E virus ORF2 protein

[0028] 1. Preparation method of ORF2 protein

[0029] The method comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying the recombinant ORF2 protein.

[0030] a. Construct expression vector:

[0031] The positive clone plasmid pMD18-T-ORF2 and the expression vector pET28a vector were digested with EcoRI and NotI, respectively, and the products were subjected to 1.2% agarose gel electrophoresis, and then recovered with a DNA gel recovery kit to obtain about 5400bp and 850bp fragments, respectively. The pET28a expression vector was constructed by directional ligation at 16°C; after the sequence and reading frame were verified by sequencing, the plasmid was linearized and transformed into BL21 competent cells.

[0032] b. Construction of expression strains, protein extraction and purification:

[0033] After tran...

Embodiment 3

[0035] Embodiment 3: the preparation of subunit vaccine

[0036] 1. Subunit vaccine preparation

[0037] 1. Preparation of ORF2 protein for seedling production

[0038] The BL21-ORF2 strain was inoculated in LB liquid medium containing kanamycin, cultured with shaking at 30°C for 18 hours, quantitatively distributed, and after pure inspection, it was used as the first-class seed. Take the first-grade seeds and inoculate them in LB liquid medium, culture them with shaking at 37°C for 18 hours, and store them at 2-8°C after microscopic examination. Add LB liquid medium according to 60% (V / V) of the volume of the fermenter, and at the same time add antifoaming agent according to 0.1% (V / V) of the medium, pass through high-temperature steam to sterilize for 30 minutes, and wait until the temperature of the medium drops to 37°C , inoculated with secondary seed solution for ORF2 protein production, the parameters of the fermenter were set to stir speed 800r / min, temperature 37°C, ...

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Abstract

The invention provides a hepatitis E virus ORF2 protein, the amino acid sequence of which is SEQ ID NO: 2. The Hexon protein of the invention can be used for preparing a vaccine or a diagnostic reagent. A hepatitis E subunit vaccine prepared by the ORF2 protein of the invention has the characteristics of high antigen stability, high purity, strong specificity, no production of other unrelated antibodies and convenient and accurate detection method. The invention has laid a solid foundation for the industrial production of the hepatitis E subunit vaccine.

Description

technical field [0001] The invention belongs to the technical field of vaccine protein screening, in particular to a hepatitis E ORF2 protein. Background technique [0002] Hepatitis E is an infectious disease caused by hepatitis E virus (Hepatitis E Virus, HEV), mainly transmitted through the intestinal tract, and has the clinical and epidemiological characteristics of acute viral hepatitis. Viral hepatitis that seriously endangers human health. Hepatitis E is currently prevalent in my country and many developing countries, and sporadic in a few developed countries. HEV is a zoonotic virus. It has been reported that HEV can infect many animals such as chicken, deer and rabbit under natural conditions. Hepatitis E can reduce egg production by more than 20% in some fulminant flocks. The HS syndrome mortality rate of laying broiler breeders and laying hens aged 30 to 72 weeks was higher than normal, and the incidence rate was highest at 40 to 50 weeks of age. Sick chickens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/51G01N33/68G01N33/576A61K39/29A61P31/14
CPCA61K39/12A61P31/14C12N2770/28134C07K14/005G01N33/6893G01N33/5767C12N2770/28122G01N2333/08
Inventor 郭伟伟向银辉蔡联燊刘新文刘义范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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