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PRRSV-1 virus-like particles and preparation method thereof

A PRRSV-1, virus-like technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viral peptides, etc., can solve the problems of unsatisfactory vaccines, large-scale production, and complicated process of virus-like particles

Inactive Publication Date: 2019-05-07
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] For this reason, the embodiment of the present invention provides a PRRSV-1 virus-like particle to solve the problems in the prior art that the process of preparing virus-like particles is complicated, cannot be produced on a large scale, and cannot meet the needs of existing vaccines for virus-like particles

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  • PRRSV-1 virus-like particles and preparation method thereof
  • PRRSV-1 virus-like particles and preparation method thereof
  • PRRSV-1 virus-like particles and preparation method thereof

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Embodiment 1

[0038] The design of the M gene sequence primer of embodiment 1 expression structural protein GP5 and matrix protein

[0039] Based on the published PRRSV LV strain (GenBank accession number: M96262.2) envelope structural protein GP5 and the nucleotide sequence of expression gene of matrix protein M, and design primers respectively for gene amplification; In matrix protein M gene The syn21 sequence is added to the upstream amplification primer as shown in SEQ ID NO.5, and the Flag sequence is added to the downstream amplification primer as shown in SEQ ID NO.6; the amplification primers are synthesized by Jilin Kumei Biotechnology Co., Ltd. The sequences and numbers of the primers for each gene amplification are as follows:

[0040] (1) The amplification primer sequence of GP5 gene is:

[0041] SEQ ID NO.1:

[0042] pGP5-F:5'-CTCGAGATGAGATGTTCTCACAAATTG-3';

[0043] SEQ ID NO.2: pGP5-R:5'-GGTACCCTAGGCCTCCCATTGCTC-3'; wherein, the 5'-end restriction site of the amplified pro...

Embodiment 2

[0050] Amplification and cloning of embodiment 2GP5 gene and M gene

[0051] Take 200 μL of PRRSV virus liquid, use the total RNA extraction kit to extract the total RNA in the virus liquid, and reverse transcribe it into cDNA. Using the cDNA as a template, using pGP5-F / pGP5-R and pM-F / pM-R in Example 1 as primers, set the reaction conditions according to the instructions of the gene synthesis report, and PCR amplify the GP5 gene and the M gene.

[0052] PCR products were separated and purified by 1% agarose gel electrophoresis. Mix 7 μL of the purified PCR product with 1 μL of pMD-19T Simple vector, 1 μL of T4 DNA ligase, and 1 μL of Buffer. In freshly thawed Trans5α competent cells, ice-bath for 20 minutes, heat shock at 42°C for 60 seconds, immediately ice-bath for 5 minutes, then activate at 37°C for 5 minutes, spread evenly on the LB culture plate containing ampicillin (50 μg / mL), 37 Cultivate upside down at ℃ for 12 hours, pick a single colony into LB liquid medium cont...

Embodiment 3

[0054] The construction of embodiment 3 recombination shuttle plasmids

[0055] The schematic diagram of the construction of the recombinant shuttle plasmid pFBD-GP5-M is as follows: figure 1 As indicated, the 19T-GP5 plasmid and the carrier plasmid were digested with restriction endonucleases XhoI and KpnI, and the GP5 gene was cloned into the downstream of the Pp10 promoter of the pFastBac Dual vector to construct the recombinant plasmid pFBD-GP5; the restriction endonuclease The 19T-M plasmid and the recombinant plasmid pFBD-GP5 were digested with enzymes BamHI and HindIII, the M gene was cloned downstream of the PpH promoter, and the recombinant shuttle plasmid pFBD-GP5-M was constructed and identified by enzyme digestion. The electrophoresis of pFBD-GP5-M is as follows figure 2 As shown, the recombinant plasmid construction and extraction methods are the same as in Example 2.

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Abstract

The embodiment of the invention discloses PRRSV-1 virus-like particles. The particles comprise PRRSV LV strain structural protein GP5, PRRSV LV strain matrix protein M, expressed PRRSV LV matrix protein M and co-expressed PRRSV LV structural protein GP5 of the virus-like particles. The embodiment further provides a method for preparing the PRRSV-1 virus-like particles. The method comprises the following steps: respectively cloning genes expressing GP5 protein and matrix protein M in a PRRSV LV strain; cloning the gene expressing the GP5 protein and matrix protein M to a baculovirus shuttle vector to acquire a recombinant baculovirus shuttle vector; and transforming the recombinant baculovirus shuttle vector to a DH10Bac competent cell to acquire a recombinant baculovirus genome. The methodis suitable for large-scale amplified preparation of anti-European porcine reproductive and respiratory syndrome vaccines, and has high safety and excellent development and application prospect.

Description

technical field [0001] The embodiment of the present invention relates to the technical field of biopharmaceuticals, in particular to a PRRSV-1 virus-like particle, a preparation method and its application. Background technique [0002] At present, porcine reproductive and respiratory syndrome (PRRS) caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) is one of the important viral infectious diseases in the pig industry. Infected pigs are mainly manifested as reproductive disorders of sows, respiratory disorders of piglets and high mortality of weaned piglets. After infection, recovered pigs are often in a state of recessive infection and can shed the virus for a long time. PRRSV is ubiquitous in the global pig industry, which has brought huge economic losses to the global pig industry, and also increased certain risks to food safety. [0003] PRRSV is a single-stranded positive-sense RNA virus with a ge...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/866A61K39/12A61P31/14
Inventor 金宁一李昌许汪杜寿文田明尧鲁会军李霄姜宇航宋利娜陈竞付婷婷郝鹏飞
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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