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A method for preparing gosling plague virus-like particles using Escherichia coli system

A gosling plague virus and virus-like technology, applied in the biological field, can solve problems such as affecting the application of VP2 protein, inability to obtain gosling plague virus-like particles, etc., and achieve the effect of good reactogenicity

Active Publication Date: 2020-06-09
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is that the gosling plague virus VP2 protein expressed by Escherichia coli mostly exists in the form of insoluble inclusion bodies, so it is impossible to obtain gosling plague virus-like particles, which affects the application of the VP2 protein expressed by Escherichia coli in vaccine research and development technical defect, providing a method for expressing gosling plague virus VP2 protein soluble in Escherichia coli, and using the soluble VP2 protein obtained by this method to prepare gosling plague virus-like particles

Method used

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  • A method for preparing gosling plague virus-like particles using Escherichia coli system
  • A method for preparing gosling plague virus-like particles using Escherichia coli system
  • A method for preparing gosling plague virus-like particles using Escherichia coli system

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Embodiment 1

[0035] Example 1 Construction and Identification of Prokaryotic Expression Recombinant Plasmid pET-Sumo-VP2

[0036] 1. Optimization of codons of goose parvovirus VP2 protein gene

[0037] Site-directed mutagenesis included mutation of codons AGA to CGC and GGA to GGT. Further, the sites of site-directed mutation include the following amino acid residue sites in goose parvovirus VP2: 39th, 40th, 57th, 58th, 157th, 257th, 403rd, 483rd bit and the 487th bit.

[0038] 2. Primer design: According to the VP2 gene sequence (AY506547) of the gosling plague virus structural protein in the NCBI gene bank and the restriction site of the pET-Sumo vector, a pair of primers with restriction sites were designed using OLigo6.0 software (provided by Shanghai Synthesized by Jierui Biotechnology Co., Ltd.), the primer sequences are as follows:

[0039] VP2-F: 5' CG GGATCC ACGGCACCCGTCAA 3' (underlined as Bam H I restriction site)

[0040] VP2-R: 5'CCC AAGCTT TCATTACAGATTTTGAGTTAGATAT...

Embodiment 2

[0110] Example 2: Expression of soluble VP2-pET-Sumo recombinant protein and determination of optimal induction conditions

[0111] First, the two recombinant plasmids (before and after VP2 gene optimization) were respectively transformed into the expression strain competent BL21 (DE3) plyss, and the positive colonies were picked after drawing the plate, and inoculated at a ratio of 1:50 (containing 100 μg ampicillin / ml) in LB liquid medium, cultured in a shaker at 37°C at 200r / min, and when the OD600 value of the bacterial solution was about 0.6, IPTG was added to a final concentration of 1mM, and the expression was initially induced at 30°C for 5 hours. For the sample, the sample was centrifuged at 4000r / min for 15 min, and the bacterial pellet was resuspended with 1 / 20 of the original volume of PBS (pH 7.4). The supernatant after sonication was subjected to SDS-PAGE according to conventional methods, and the recombinant protein expression levels before and after optimizati...

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Abstract

The invention relates to a method for preparing gosling plague virus-like granules with an escherichia coli system for soluble expression of gosling plague virus VP2 protein. The method for soluble expression of gosling plague virus VP2 protein comprises the following steps: performing codon optimization on a gosling plague virus VP2 gene, performing site-specific mutagenesis, namely, mutating a codon AGA into CGC and mutating GGA into GGT, cloning to a pET-Sumo vector, establishing a recombinant expression vector pET-Sumo-VP2, transforming the pET-Sumo-VP2 into a prokaryotic expression bacterium, and inducing with IPTG (isopropyl beta-D-1-Thiogalactopyranoside) at 37 DEG C so as to obtain soluble recombinant VP2 recombinant protein; and performing digestion on the recombinant protein with a ULP enzyme, and purifying with a Ni column, thereby obtaining purified VP2 protein. Electron microscope results show that the gosling plague virus-like granules can be prepared from VP2 protein after digestion, and moreover, the purified VP2 protein has good reactogenicity and can be applied to preparation of subunit vaccines of gosling plague virus genetic engineering.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for preparing gosling plague virus-like particles by soluble expression of gosling plague VP2 protein in an Escherichia coli system. Background technique [0002] Goose parvovirus parvovirus , GPV) Infection at the beginning, scholars from different countries called it goose flu, also known as goose plague or goose plague, goose enteritis, foie hepatitis, goose infectious myocarditis, etc. The diversity of the disease name reflects the diversity of its pathological features and the severity of harm to susceptible animals. Goslings of different ages will have different clinical symptoms after being infected with the virus, which are manifested as chronic type, subacute type and acute type, and the course of the acute type can lead to 100% death of infected geese. [0003] Gosling plague virus (GPV) belongs to the family Parvoviridae, the virion has no envelo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/35C12N15/70C12N7/04C07K14/015C07K16/08A61K39/23A61P31/20
CPCA61K39/00C07K14/005C07K16/081C12N7/00C12N15/70C12N2750/14322C12N2750/14323C12N2750/14334C12N2800/101C12N2800/22Y02A50/30
Inventor 曲光刚沈志强金婷婷李书光武曰星王长江
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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