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Soluble recombinant protein and expression and purification methods and application thereof

A recombinant protein and expression method technology, applied in the field of recombinant proteins, can solve the problems of unsuitable large-scale production, complex preparation process, poor immunogenicity, etc., and achieve the effects of being suitable for large-scale industrial production, improving the purity, and being easy to operate.

Inactive Publication Date: 2017-08-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to solve the technical problems of the existing PCV2 virus-like particle vaccines, such as complex preparation process, high cost, small processing capacity, poor immunogenicity, and unsuitability for large-scale production. Therefore, the present invention provides a soluble recombinant protein, The present invention also provides the expression method and purification method of the soluble recombinant protein, and the present invention also provides the purposes of the soluble recombinant protein in preparing PCV2 virus-like particle vaccine

Method used

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  • Soluble recombinant protein and expression and purification methods and application thereof
  • Soluble recombinant protein and expression and purification methods and application thereof
  • Soluble recombinant protein and expression and purification methods and application thereof

Examples

Experimental program
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Embodiment 1

[0035] Embodiment 1: Construction of the soluble recombinant protein expression vector of the present invention

[0036] 1.1 Acquisition of Cap protein gene

[0037] Based on the sequences of PCV2 epidemic strains in the NCBI database, after comparison, the Cap protein amino acid sequence of a epidemic strain was selected, which was identical or highly similar to the corresponding sequences of other epidemic strains. As shown in SEQ ID NO:1. Under the premise of ensuring that the amino acid of the Cap protein of this strain remains unchanged, the common codons of Escherichia coli are used to translate the amino acid sequence into a nucleotide sequence, and the nucleotide sequence is shown in SEQ ID NO: 2, making it suitable for use in Escherichia coli Expression, and then entrust Jinweizhi Company to carry out gene synthesis. During gene synthesis, restriction sites are added at both ends. The nucleotide sequence after adding restriction sites is as SEQ ID NO: 3, and the synt...

Embodiment 2

[0042] Embodiment 2: Expression and purification of the soluble recombinant protein of the present invention

[0043] 2.1 Mass expression of soluble recombinant protein

[0044] (1) Take out the Escherichia coli strain carrying the recombinant plasmid pET-28a-Cap-gene from -20°C, insert it into kanamycin sulfate-resistant LB liquid medium at a ratio of 1:1000, and cultivate overnight on a shaker at 220rpm at 37°C (12-14h).

[0045] (2) Calibrate the PH electrode of the 5L fermentation tank, prepare 2.5L medium and put it into the tank, sterilize at 121°C for 20 minutes, connect the automatic control system after cooling, correct the dissolved oxygen, adjust the ventilation and the initial speed.

[0046] (3) 10% seed liquid is connected to the fermenter, the temperature is 37°C, the stirring speed and ventilation are manually adjusted, and the dissolved oxygen is controlled between 30% and 60%.

[0047] ⑷ When the dissolved oxygen starts to rise rapidly, add the feeding medi...

Embodiment 3

[0061] Embodiment 3: the preparation of PCV2 virus-like particle vaccine of the present invention

[0062] First, use the BCA kit to measure the above-mentioned purified soluble recombinant protein, adjust the concentration of the soluble recombinant protein to 1mg / ml, and then mix the protein with 201 adjuvant provided by Sepic company in France according to the volume ratio of 1:1, and then carry out Phacoemulsification. Store at 4°C after testing the viscosity and stability.

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Abstract

The invention provides a soluble recombinant protein. The soluble recombinant protein is obtained through prokaryotic expression of a gene of a porcine circovirus II type capsid protein. The amino acid sequence of the porcine circovirus II type capsid protein is shown in SEQ ID NO:1. During prokaryotic expression of the Cap protein, a gene for expressing the Cap protein is optimized through a codon, so that soluble expression of the Cap protein in escherichia coli can be improved remarkably. Target protein is purified with a two-step gradient salting-out precipitation method, operation is easy, purity is improved greatly, the production cost is low, the immunogenicity of the obtained protein is high, and large-scale industrial production can be achieved. A PCV2 virus-like particle vaccine does not contain viral nucleic acid and has high safety. The prepared PCV2 virus-like particle vaccine has no pathogenicity to experiment animals, and after animal immunization, a porcine circovirus antibody can be generated quickly at a high level, and lasting time is long.

Description

technical field [0001] The invention relates to a recombinant protein, in particular to a soluble recombinant protein and its expression and purification method and use. Background technique [0002] Porcine circovirus 2 (PCV2) is the main pathogen of postweaning multisystemic wasting syndrome (PMWS) in piglets. Since PMWS was first reported in Canadian pigs in 1991, the disease has spread worldwide Popularity. At present, vaccination is an effective means to prevent and control PCV2 infection. Vaccines that have been commercialized at home and abroad mainly include whole-virus inactivated vaccines and subunit vaccines. Although whole-virus inactivated vaccines have been produced, they need to be concentrated sometimes because of their weak value-added ability in vitro culture and low virus titers. Meet the requirements, and there are problems such as difficult to unify the quality between batches in production. In contrast, subunit vaccines have better safety and high im...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/01C12N1/21A61K39/12A61P31/20C12R1/19
CPCA61K39/12A61K2039/5258A61K2039/552C07K14/005C12N15/70C12N2750/10022C12N2750/10034C12N2800/22
Inventor 樊惠英刘洁谢永生
Owner SOUTH CHINA AGRI UNIV
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