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Pig circovirus III-type virus-like particle and preparation method thereof

A porcine circovirus and virus-like technology is applied in the fields of botanical equipment and methods, biochemical equipment and methods, viruses, etc. It can solve the problems of poor immunogenicity of virus-like particles and is not suitable for large-scale production, and achieve good development and Application prospect, high safety, good immunogenic effect

Active Publication Date: 2019-09-06
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For this reason, the embodiment of the present invention provides a porcine circovirus type III virus-like particle and its preparation method to solve the problems that the virus-like particles prepared in the prior art have poor immunogenicity and are not suitable for large-scale production

Method used

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  • Pig circovirus III-type virus-like particle and preparation method thereof
  • Pig circovirus III-type virus-like particle and preparation method thereof
  • Pig circovirus III-type virus-like particle and preparation method thereof

Examples

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Effect test

Embodiment 1

[0025] Example 1 Design and synthesis of porcine circovirus type III Cap protein gene

[0026] According to the published nucleotide sequence of porcine circovirus type III Cap protein (GenBank accession number: KX966193.1), add EcoRI enzyme before the start codon ATG of porcine circovirus type III Cap protein nucleotide sequence Cutting site: gaattc, add a His tag sequence after ATG, as shown in SEQ ID NO.2, add a Not I restriction site after the stop codon: gcggccgc, the optimally designed Cap protein gene sequence is shown in SEQ ID NO.1 Show. The sequence of the forward primer PCV3F for amplification of the nucleotide sequence of the Cap protein gene is shown in SEQ ID NO.3; the sequence of the reverse primer PCV3R is shown in SEQ ID NO.4. The gene was synthesized by Jinweizhi Biotechnology Co., Ltd., and cloned into the pUC57 vector, named pUC57-Cap.

Embodiment 2

[0027] Example 2 Construction and Identification of Recombinant Shuttle Plasmid pFB-Cap

[0028] Use restriction endonucleases EcoRI and NotⅠ to double digest pUC57-Cap and pFastBac 1 plasmids, react at 37°C for 2 hours, use 1% agarose gel electrophoresis to separate, and recover the Cap protein gene fragment and linearized pFastBac 1; Add 1 μL each of T4DNA ligase, T4Buffer, and linearized pFastBac 1 to 7 μL of the Cap protein gene fragment, mix gently, incubate at 25°C for 10 minutes, add the mixture to the just-thawed Trans5α competent cells, and incubate on ice for 20 minutes , heat shock at 42°C for 45s, immediately ice-bathed for 5min, activated at 37°C for 5min, spread evenly on the LB culture plate containing ampicillin (50μg / mL), culture upside down at 37°C for 12h, and obtain recombinant shuttle plasmid pFB-Cap colonies .

[0029] Pick a single colony, inoculate it in liquid LB medium containing ampicillin (50 μg / mL), culture at 37 °C, 220 rpm for 12 h with shaking,...

Embodiment 3

[0030] Example 3 Construction of rB-Cap recombinant bacmid and extraction of rB-Cap recombinant bacmid

[0031] Take 1ng of the recombinant shuttle plasmid pFB-Cap in Example 2 and add it to the DH10Bac competent cells that have just been thawed on ice, mix gently and bathe in ice for 20min, heat shock at 42°C for 45s, immediately bathe in ice for 5min, use SOC without antibiotics The medium was shaken and activated at 37°C for 4 hours, and 80 μL was evenly spread on the medium containing kanamycin (50 μg / mL), tetracycline (10 μg / mL), gentamicin (7 μg / mL), X-Gal (100 μg / mL) , IPTG (40 μg / mL) on the LB blue-white screening plate, cultured upside down at 37°C for 48 hours, picked white colonies for PCR identification, and used the three-line method to streak the bacterial solution containing the target band on the above-mentioned LB blue-white screening plate , after inverting at 37°C for 36 hours, pick the white colony for PCR identification to obtain the rB-Cap recombinant vec...

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Abstract

An embodiment of the invention discloses a pig circovirus III-type virus-like particle. A preparation method of the virus-like particle includes the steps: amplifying pig circovirus III-type Cap protein genes; constructing recombinant shuttle-plasmid pFB-Cap by the aid of the genes; constructing rB-Cap recombinant rod granules by the aid of the recombinant shuttle-plasmid pFB-Cap; transfecting therB-Cap recombinant rod granules into SF9 cells to obtain recombinant baculovirus rBV-PCV3 Cap expressing pig circovirus III-type Cap genes; enabling the recombinant baculovirus rBV-PCV3 Cap to infectHigh Five cells, and purifying the recombinant baculovirus rBV-PCV3 Cap infected by the High Five cells to obtain the pig circovirus III-type virus-like particl PCV3 VLP. A nucleotide sequence of theCap protein gene is as shown in SEQ ID NO.1. According to the particle, based on a baculovirus-insect cell expression system, preparation is implemented by the aid of High Five cell expression of serum-free suspension culture, and the PCV3 virus-like particle is acquired by combining sucrose cushion ultracentrifugation and sucrose density gradient centrifugation purification. The virus-like particle is good in immunogenicity and high in safety and has good development and application prospects.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of biopharmaceuticals, in particular to a method for preparing porcine circovirus type III virus-like particles. Background technique [0002] Clinically, pigs infected with porcine circovirus type Ⅲ (porcine circovirus 3, PCV3) often have dark spots on the abdomen, stillbirths or mummified peptides, but the feed intake and mental state of breeding pigs are good; mixed infections often occur in nursery pigs, Extremely poor mental state, high fever, abdominal respiration, etc., the mortality rate of pig farms with severe disease exceeds 15%, which has brought certain economic losses to the breeding industry. [0003] PCV3 is a single-strand circular DNA virus with a genome length of about 2.0kb, a virus ion diameter of about 17-20nm, and no envelope. The amino acid homology of the Cap protein of PCV3 and PCV2 is only 30%, and the Cap protein is the main protein for PCV to induce spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/866A61K39/12A61P31/20A61P17/00A61P13/12A61P29/00
CPCA61K39/12A61K2039/552A61P13/12A61P17/00A61P29/00A61P31/20C07K14/005C12N15/86C12N2710/14043C12N2750/10023C12N2750/10034
Inventor 金宁一李昌许汪杜寿文王茂鹏田明尧鲁会军李霄郝鹏飞宋利娜陈竞姜宇航
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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