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Recombined hepatitis E hepatitis virus protein, preparation method and usage thereof

A hepatitis E virus and polynucleotide technology, applied in the fields of molecular biology and medicine, can solve the problems of unsuccessful development and achieve good antigenicity and immunogenicity, strong pertinence and practical value

Inactive Publication Date: 2007-10-31
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there have been no reports of successful development so far

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  • Recombined hepatitis E hepatitis virus protein, preparation method and usage thereof
  • Recombined hepatitis E hepatitis virus protein, preparation method and usage thereof
  • Recombined hepatitis E hepatitis virus protein, preparation method and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This implementation describes the construction method of recombinant bacteria BL21(DE3)-pET-28a(+)-179:

[0051] (a) Obtaining the target gene: Using the sequence of the Chinese strain of HEV genotype 4 as a template, use primer 1 (5'-CCCCCCATGGTTATCCAGGACTATGATAATC-3') and primer 2 (5'-CCCCTCGAGTCAAGGGTAATCAACAGTGTCCTCCA-3') to amplify the ORF2 encoded polypeptide 453 -631 (p179) gene fragment. The PCR conditions are: 94°C-45 seconds, 52°C-45 seconds, 72°C-50 seconds, 35 cycles. Among them, primer 1 and primer 2 respectively contain NcoI and XhoI enzyme cutting sites. PCR products were recovered and purified by agarose gel.

[0052] The above amino acid sequence from position 453 to position 631 (p179), the amino acid sequence described in it is as follows:

[0053] VIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPM

[0054] YVSDTVTFVNVATGAQGVRSRSLDWSKVTLDGRPLTTIQQYSKTFYVLPLRGKLS

[0055] FWEAGTTKAGYPYNYNTTASDQILIENAAGHRVCISTYTTNLGSGPVSISAVGVL

[0056] APHSAL...

Embodiment 2

[0073] This implementation describes the purification method of recombinant p179 protein and its electron microscope analysis results (Fig. 6-Fig. 8):

[0074] Purification of the recombinant p179 protein was carried out by the following method: the recombinant strain BL21(DE3)-pET-28a(+)-179 expressed the target protein under the induction of IPTG at 37°C. The bacteria were collected by centrifugation at 5000g at 4°C for 15 minutes, and the bacteria were resuspended in buffer A (20mM Tris-HCl, pH 7.4), and the bacteria were lysed by ultrasonic method. Then, the precipitate was removed by centrifugation at 20,000 g at 4°C for 30 minutes. It should be noted that HEV p179 is a non-fusion protein and mostly exists in the supernatant of the lysate in a soluble form. HEV p179 was purified using Biologic LP System (Bio-Red, England). First, add the supernatant obtained from the lysis to an anion exchange chromatography column (SOURCE 30Q, 10ml bed volume) that has been pre-balanced...

Embodiment 3

[0077] This implementation describes the evaluation of recombinant p179 protein as an antigen for the indirect ELISA detection of HEV IgG antibody, wherein the coating dose range of recombinant p179 protein is 20-80ng / well, and the preferred concentration is 50ng / well.

[0078] (a) Detection steps:

[0079] Coat polystyrene microwell strips (50ng / well) with recombinant p179 protein as an antigen in advance, overnight at 4°C; wash the plate 3 times with 1×PBST washing solution the next day, and then dry it; seal the pre-coated microwell plate with blocking solution , 37°C for 1h; washed twice with PBST, button-dried; sealed, and stored at 4°C for later use.

[0080] Adding samples: add 95 μl sample diluent to each well, add 5 μl serum samples in turn, set up blank control wells, negative control serum, positive control serum and samples to be tested, and pat to mix;

[0081] Incubation: incubate at 37°C for 30 minutes;

[0082] Washing: Discard the liquid in the well, wash 5 ...

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Abstract

The invention discloses a restructuring the fifth of the ten Heavenly Stems type hepatitis virus protein, coded sequence and usage, which is characterized by the following: the HEV virus strain to prepare restructuring the ten Heavenly Stems vaccine is the first gene type; the HEV p179 is based on popular production of the forth gene type HEV virus strain and possesses stronger placement and practical value; the HEV p179 can get highly effective expression in pronucleus system and 90% protein is nature solubility; the HEV p179-VLPs with diameter at about 20nm is constituted by 179aa; the HEV p179-VLPs is smallest restructuring HEV protein compared to now eucaryon system and HEV VLPs of pronucleus system expression.

Description

1. Technical field [0001] The invention belongs to the field of molecular biology and medicine, in particular to a recombinant hepatitis E virus protein, its preparation method and application. 2. Background technology [0002] Prior Art: Hepatitis E virus (HEV) is the main pathogen causing acute infectious hepatitis in Southeast Asia, Central Asia, and Africa. HEV is mainly transmitted through the fecal-oral route, and is usually caused by drinking contaminated water sources. The epidemic modes of HEV caused by HEV mainly include outbreaks and distribution. HEV infection can affect patients of different ages, but infection is more common in adults. Generally speaking, the clinical symptoms of hepatitis E and hepatitis A are similar. Current studies believe that the infection rate of HEV has exceeded that of hepatitis A in some developing countries, and the condition is more serious. Studies have shown that among all types of hepatitis, E The liver has the highest mortalit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/51G01N33/68G01N33/576C12Q1/68A61K39/29A61P1/16
Inventor 孟继鸿戴星董晨
Owner SOUTHEAST UNIV
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