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Genetic engineering subunit vaccine for porcine circovirus as well as preparation method and application of genetic engineering subunit vaccine

A porcine circovirus and subunit vaccine technology, applied in vaccines, antiviral agents, veterinary vaccines, etc., to achieve the effects of reduced virus content, simple preparation process, and high purity

Pending Publication Date: 2018-10-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing PCV2 vaccine is ineffective against PCV3. Therefore, the research and development of PCV3 vaccine is imminent.

Method used

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  • Genetic engineering subunit vaccine for porcine circovirus as well as preparation method and application of genetic engineering subunit vaccine
  • Genetic engineering subunit vaccine for porcine circovirus as well as preparation method and application of genetic engineering subunit vaccine
  • Genetic engineering subunit vaccine for porcine circovirus as well as preparation method and application of genetic engineering subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Expression of recombinant Cap protein by E. coli

[0043] 1. Gene Amplification

[0044] Amplify Cap gene with PCV3-China / GD2016 virus strain (PCV3-China / GD2016 genome information has been disclosed in Genomecharacterization of a porcine circovirus type 3 in South China, Changxu Song, Transbound Emerg Dis. 2017 Mar 13. in the paper) as template, The reaction system is shown in Table 1, and the reaction program is shown in Table 2.

[0045] Table 1 Reaction system for Cap gene amplification

[0046]

[0047] Among them, the upstream primer is: 5'-GGCggatccATGAGACACAGACCTATATTC-3', the downstream primer is: 5'-GGGctcgagGAGAACTGACTTGTAACGAAT-3', the lowercase letters indicate the restriction sites BamHI and XhoI respectively.

[0048] Table 2 PCR reaction program for Cap gene

[0049]

[0050] (3) Gel electrophoresis of PCR amplification products

[0051] Referring to the method of "Molecular Cloning Experiment Guide", prepare a 1.2% agarose gel, take 5 μ...

Embodiment 2

[0120] Example 2 Baculovirus expresses recombinant Cap protein

[0121] 1. Gene Amplification

[0122] Same as Example 1.

[0123] 2. Cap target fragment gel recovery

[0124] Same as Example 1.

[0125] 3. Cap fragment and pFastBac-HTB double digestion

[0126] The obtained Cap gene fragment and pFastBac-HTB plasmid were digested with BamHI and XhoI, respectively, and the digestion systems are shown in Table 6 and Table 7.

[0127] Table 6 Double digestion reaction system of pFastBac-HTB plasmid

[0128]

[0129] Table 7 Double enzyme digestion reaction system of Cap gene fragment

[0130]

[0131] Incubate in a metal bath at 37°C for 20 min. After the reaction is completed, the digestion product is analyzed by agarose gel electrophoresis according to the molecular cloning manual, and the target fragment is recovered. For the recovery method, refer to the instructions of the gel recovery kit (Omega company).

[0132] 4. Cap fragment and pFastBac-HTB ligation

[0...

Embodiment 3

[0189] Embodiment 3 Vaccine immune effect experiment

[0190] Fifteen healthy susceptible piglets about 3 weeks old, without PCV3 antigen and antibody, without PRRSV, PEDV, PRV, Staphylococcus suis and other major swine pathogens, were selected and randomly divided into 3 groups. The piglets were injected after mixing equal volumes of immunogen and adjuvant according to Table 10 before injection. 3 weeks after immunization, serum was collected to determine Cap-specific antibody titers; 1 week after blood collection, challenge was performed. The challenge strain was PCV3-China / GD2016, and the titer was 10^ 6 TCID50, 1 mL per head, intranasally, 14 days after challenge, lymph nodes were collected, and the content of PCV3 in the tissue was determined by fluorescence quantitative PCR.

[0191] Table 10 Immune effect experiments

[0192]

[0193] The specific operation of the fluorescent quantitative PCR method is as follows: Design quantitative primers for the Cap protein ge...

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Abstract

The invention discloses a genetic engineering subunit vaccine for a porcine circovirus as well as a preparation method and application of the genetic engineering subunit vaccine. By cloning the nucleocapsid protein of the novel porcine circovirus 3 (PCV3), a PCV-Cap protein with higher purity is successfully expressed by using an escherichia coli or baculovirus expression system. The subunit vaccine for the PCV3 is successfully developed for the first time by using the PCV-Cap protein; the prepared vaccine is high in antigen purity, good in safety and strong in immunogenicity, and has no pathogenicity to pigs and other animals; the antigen has good solubility in a neutral PH buffer solution; furthermore, the preparation method is simple and low in cost, thus being suitable for large-scaleindustrial production; an effective and powerful means is provided for the prevention and control of novel PCV, and the genetic engineering subunit vaccine has a wide application prospect in the fieldof the prevention and control of the PCV3.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a porcine circovirus genetically engineered subunit vaccine and a preparation method and application thereof. Background technique [0002] Circovirus belongs to the genus Circovirus of the family Circoviridae, and its genome is a circular single-stranded DNA of about 2 kb. Circoviruses can infect a variety of animals, including pigs, dogs, ducks, chickens, foxes, and fish. Circovirus mainly encodes two open reading frames (rep and cap). Two circoviruses are known to infect pigs. Porcine circovirus type 2 (PCV2) infection can cause different clinical diseases and cause severe economic losses to the pig industry, while circovirus type 1 (PCV1) does not cause clinical disease. In 2015, American researchers detected a novel circovirus (PCV3) in the United States through metagenomic sequencing, which is associated with dermatitis-nephrotic syndrome, reproductive disorde...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/39A61P31/20
CPCA61K39/12A61K39/39A61K2039/552A61K2039/55505
Inventor 宋长绪王磊刘业兵申翰钦张乐宜刘艳玲梁鹏帅张鹏飞刘相聪
Owner SOUTH CHINA AGRI UNIV
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