A kind of porcine circovirus type ii genetic engineering subunit vaccine and its application

A porcine circovirus, virus-like technology, applied in the direction of genetic engineering, application, viral peptides, etc., can solve the problems of cost reduction and popularization and application, high process cost, unfavorable vaccines, etc., and achieve low cost, broad application prospects, and antigen purity high effect

Active Publication Date: 2021-11-02
兆丰华生物科技(南京)有限公司北京生物医药科技中心 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing PCV2 recombinant subunit vaccines are expressed in insect cell systems. The recombinant proteins produced by this process can form natural VLPs and have good immunogenicity. However, the cost of this process is relatively high, which is not conducive to reducing the cost of the vaccine and promoting its application.

Method used

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  • A kind of porcine circovirus type ii genetic engineering subunit vaccine and its application
  • A kind of porcine circovirus type ii genetic engineering subunit vaccine and its application
  • A kind of porcine circovirus type ii genetic engineering subunit vaccine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Sequence Synthesis

[0024] Through sequence comparison, the nucleotide sequence was changed under the premise of ensuring the same encoded amino acid, the rare codons in the sequence were replaced with E. coli preferred codons, and a 6His or 8His tag and a stop codon were added to the 3' end of the PCV2ORF2 gene sequence The 5' end of the sequence is the Nde I restriction endonuclease recognition site, and the 3' end is the Xho I restriction endonuclease recognition site. All the above sequences are artificially synthesized sequences. The synthetic sequence was digested with Nde I and Xho I and connected to the pET-21a(+) vector (see figure 1 ).

Embodiment 2

[0025] Example 2 Construction of expression vector pET-21a-PCV2-ORF2

[0026] Digest the recombinant plasmid with restriction endonucleases Nde I and Xho I, separate the digested products by agarose gel electrophoresis, and recover small fragments by cutting the gel (see figure 2 ), and then subcloned into the linearized vector pET-21a(+), the vector linearization process uses the same restriction endonuclease, the connection solution is transformed into Escherichia coli DH5α, and the positive clones are screened and verified by sequencing. After the frames are correct, the recombinant vectors are named pET-21a-PCV2-ORF2-6 His and pET-21a-PCV2-ORF2-8His respectively, and transformed into BL21(DE3) competent cells for protein expression.

Embodiment 3

[0027] Induced expression of embodiment 3 recombinant protein

[0028] (1) Pick the recombinant expression strains pET-21a-PCV2-ORF2-6His and pET-21a-PCV2-ORF2-8His and inoculate scattered single colonies in LB liquid medium containing ampicillin, and shake overnight at 37°C .

[0029] (2) Transfer the positive BL21 bacterial solution to the LB liquid medium containing Amp at a ratio of 1:100, culture it with shaking at 37°C until the logarithmic growth phase (OD600 reaches 0.6-0.8), take 100μl sample in In a sterile Eppendorf tube as a pre-induction control;

[0030] (3) Add IPTG with a final concentration of 1.0mmol / L to the above bacterial solution to induce expression of the recombinant protein, and take samples at 3, 4, 5, 6, 7, and 8 hours after adding IPTG;

[0031] (4) Centrifuge at 12,000 rpm at 4°C for 5 minutes to collect the bacteria induced to express, resuspend in PBS, and wash twice;

[0032] (5) Discard the supernatant completely, and resuspend the bacterial...

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Abstract

The invention discloses a porcine circovirus type 2 recombinant CAP protein and a subunit vaccine of the recombinant CAP protein. The invention provides a recombinant CAP protein of porcine circovirus type 2. The C-terminus of the Cap protein is fused with 8 histidine tags, and its amino acid sequence is shown in SEQ ID No.1. Compared with the inactivated vaccine of PCV2 currently used, the porcine circovirus type 2 Cap protein subunit vaccine expressed by recombinant Escherichia coli of the present invention has a relatively simple preparation process and lower cost, so it has a wider scope in the prevention and treatment of PCV2. application prospects.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a porcine circovirus type II genetically engineered subunit vaccine and its application. Background technique [0002] Porcine circovirus disease (PCVAD) infection caused by porcine circovirus type 2 (PCV2) has spread all over the world, causing very serious economic losses. The immune function of PCV2-infected pigs is suppressed, leading to secondary infection and severe clinical disease. Effective prevention and control of PCV2 has become a serious problem faced by the pig industry in my country and the world, and vaccination is a reliable method to prevent and control PCV2 infection. [0003] There are several whole-virus inactivated vaccines on the market in my country. Although whole-virus inactivated vaccines have no pathogenicity and relatively stable biological functions, there are still potential safety problems if the inactivation is not complete. In additi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01A61K39/12A61P31/20C12N15/70C12N15/34C12N1/21
CPCA61K39/12C07K14/01A61K2300/00
Inventor 石艳丽张家龙周景云刘培培郭家明于萍萍张学贤王贵华赵亚荣
Owner 兆丰华生物科技(南京)有限公司北京生物医药科技中心
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