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A kind of recombinant porcine circovirus type 2 cap protein with dominant epitope in series and its application

A porcine circovirus, dominant epitope technology, applied in the direction of viruses, applications, and viral peptides, can solve the problems of inability to completely prevent PCV2 infection or transmission, poor protection rate, and low virus titer, and achieve good immune protection. Reduced production costs and high protein expression

Active Publication Date: 2022-03-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, four commercial vaccines have been widely used in the world, and two of them use PCV2 Cap protein as the immunogen. Experimental and field studies have clearly shown that PCV2 vaccine can reduce viremia, eliminate PCVAD, and increase growth performance, etc. However, PCVAD still occurs in vaccinated pigs, which means that the immunization of existing commercial vaccines cannot completely prevent PCV2 infection or transmission; in addition, there are low virus titers obtained from whole virus culture, Incomplete inactivation leads to the possibility of stronger virulence, high price and poor protection rate

Method used

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  • A kind of recombinant porcine circovirus type 2 cap protein with dominant epitope in series and its application
  • A kind of recombinant porcine circovirus type 2 cap protein with dominant epitope in series and its application
  • A kind of recombinant porcine circovirus type 2 cap protein with dominant epitope in series and its application

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Experimental program
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Effect test

Embodiment 1

[0054] Embodiment 1 Construction of recombinant baculovirus transfer vector

[0055] (1) Gene sequence design and synthesis of recombinant proteins

[0056] Using the complete gene sequence of PCV2 LG strain (HM038034.1) recorded in GenBank as a template, the Cap / Rep linear epitope was introduced into the PCV2 cap protein. Peptides with good antigenicity and high specificity, namely the 81-100, 201-220 on the Rep protein and the 61-85, 113-131, 169-180, 192-202 epitopes on the Cap protein, And determine the tandem sequence between the dominant antigenic epitopes, and introduce the honeybee melittin signal peptide sequence (HBM) in order to achieve secreted expression, design a base sequence encoding 6 histidines before the stop codon, and finally The nucleotide sequence of the recombinant protein PCV2-TBCap was designed, and the nucleotide sequence was codon-optimized for insect cells, and then sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis. The nucleotide se...

Embodiment 2

[0076] Example 2 Obtaining of recombinant baculovirus and optimization of recombinant protein expression conditions

[0077] (1) Obtaining recombinant bacmid

[0078] Transform the successfully identified recombinant baculovirus transfer vectors pFBD-Cap-1, pFBD-Cap-2, pFBD-TBCap-1, and pFBD-TBCap-2 into DH10Bac-competent cells respectively, and gently blow and mix with a pipette gun, and place on ice ② Take the competent cells out of the ice bath, place them in a water bath at 42°C for 45 seconds, and then take them out and incubate them in the ice bath for 2 minutes; ③Add 900 μL of SOC medium to the EP tube, and incubate at 37°C Bacteria culture shaker 200rpm shaking culture transposition 4h; ④ use SOC medium to gradiently dilute the transformed bacteria (10 -1 , 10 -2 , 10 -3 ), respectively draw 100 μL of the above-mentioned diluted gradient transformed bacteria and spread them on the solid medium of LB (Gen, Kan, Tet, IPTG, X-Gel, use concentration according to Bac-to-...

Embodiment 3

[0111] The immune efficacy experiment of the subunit vaccine prepared by embodiment 3 recombinant protein

[0112] (1) Preparation of PCV2 subunit vaccine

[0113] Inoculate the third-generation recombinant baculovirus solution Ac-Cap-2 and Ac-TBCap-2 at a concentration of 2×10 6 Cells / mL High Five suspension cells were placed in a constant temperature shaker at 27°C at 120rpm to shake the cells, and the cell pellets were collected by centrifugation according to the optimal harvest time, and an appropriate amount of PBS was added to resuspend the cells, placed on ice, and the cells were ultrasonically broken. , and then centrifuged at 11,000 rpm for 15 min at 4°C. The collected supernatant is the expressed immune antigen solution.

[0114] Adjust the concentration of the recombinant protein to 1000 μg per ml, and emulsify the recombinant protein Cap and TBCap of this concentration with the ISA201VG adjuvant at a ratio of 1:1 to prepare a PCV2 subunit vaccine containing 100 μ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a recombinant porcine circovirus type 2 Cap protein in series with dominant epitopes and an application thereof. The amino acid sequence of the recombinant porcine circovirus type 2 Cap protein of the tandem dominant epitope is shown in SEQ ID No.1, and the nucleotide sequence encoding the protein is shown in SEQ ID No.2. The present invention further constructs a dual-promoter transfer vector containing the above-mentioned nucleotide sequence, and transforms Escherichia coli to obtain a recombinant baculovirus plasmid; then transfects insect cells to obtain a recombinant baculovirus, and its recombinant protein expression reaches 914 μg / ml, can specifically combine with positive serum, and has good immunological reactivity. The subunit vaccine provided by the invention can stimulate mice to produce a high level of humoral immune response after immunization of mice; can induce a specific immune response in the body after immunization of piglets, and can provide good immune protection for challenged piglets.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant porcine circovirus type 2 Cap protein in series with dominant epitopes and an application thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes multisystemic wasting syndrome (PMWS) in weaned piglets. PCV2 mainly invades the immune system of the body, causing severe immunosuppression, which is prone to secondary or mixed infection of other pathogenic microorganisms, and seriously endangers the health of pigs. Since the advent of porcine circovirus type 2 vaccine in 2006, vaccination has become the main means of preventing and treating porcine circovirus disease. The Cap protein encoded by PCV2 ORF2 is the main structural protein of the virus and has a type-specific epitope, which can induce the body to produce a specific immune response. It is an ideal target for the development of PCV2 genetically engineer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/867C12N7/01A61K39/12A61P31/20
CPCC07K14/005C12N15/86C12N7/00A61K39/12A61P31/20C12N2710/14021C12N2710/14043C12N2800/22C12N2750/10022C12N2750/10034A61K2039/552
Inventor 陈金顶吴珂珂罗朝唯易琳赵明秋丁红星
Owner SOUTH CHINA AGRI UNIV
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