Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof

A subunit vaccine and virus technology, applied in botany equipment and methods, biochemical equipment and methods, antiviral agents, etc., can solve the problems of low virus titer, insufficient antigen content, poor biological safety, etc.

Inactive Publication Date: 2012-02-15
ZHEJIANG YEBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Production of inactivated circovirus vaccines using traditional porcine kidney cell cu

Method used

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  • Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof
  • Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof
  • Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Preparation of subunit vaccines

[0086] The insect cell Sf9-F was cultured with the suspension culture cell technology in a common cell culture device, the culture environment was 27°C 120r / min, and the cells were in the logarithmic growth phase for about 48 hours, and the recombinant baculovirus Bac / CY( CGMCCNo.5267 strain), and then through 72h culture, harvest infected cells, the virus titer can reach 10 8.0 TCID 50 / ml or more, repeated freeze-thawing 3 times, washed several times with PBS to get the supernatant, purified the above-mentioned recombinant protein, added conventional vaccine adjuvant and emulsified to obtain PCV2 recombinant baculovirus subunit vaccine.

Embodiment 2

[0088] Sf9-F cells cultured in the torrent reactor AP20C for the preparation of subunit vaccines

[0089] Sf9-F cells were inoculated into the torrent bag, and after 84 hours of culture, the number of cells reached 2.98 6 pcs / ml (attached Figure 5). After inoculating the virus seed suspension of recombinant baculovirus CGMCC No.5267 strain and continuing to culture for 6 days, the antigen expression test results showed that: in the early stage of virus infection, the antigen protein was mainly concentrated in the cell pellet, and the antigen content in the supernatant was very small; , the antigens were mainly distributed in the supernatant; the highest level of antigen expression appeared at 120 hours after inoculation, and the total antigen content could reach 238.17 μg / ml (attached Figure 6 ). Other processes are with embodiment 1.

Embodiment 3

[0091] Preparation of PCV2 Recombinant Baculovirus Subunit Vaccine

[0092] The insect cell Sf9-F can be cultivated by the spinner bottle culture cell technique to prepare the PCV2 recombinant baculovirus subunit vaccine. Other processes are with embodiment 1.

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Abstract

The invention relates to a porcin circovirus type 2 (PCV2) recombinant autographa californica multicapsid nucleopolyhedrovirus construction method and a subunit vaccine preparation method thereof. The novel recombinant lucerne fork vein noctuid nuclear polyhedrosis virus construction method comprises the steps that: the main surface antigen which expresses the PCV2 is used to prepare the subunit vaccine with better immunity. Specifically, in the carrier expression system of the PCV2 recombinant autographa californica multicapsid nucleopolyhedrovirus, a hepatitis E virus open reading frame (ORF2) gene of the PCV2 is directionally inserted, so the gene efficiently expresses in insect cells and has good immunogenicity, the prepared corresponding subunit vaccine can stimulate pigs to generate the protective immunoreaction against the attack of the strong virus of the PCV2.

Description

technical field [0001] The present invention relates to a kind of PCV2 recombinant construction and its subunit vaccine preparation method, specifically refers to a kind of PCV2 recombination S. products industry. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to Circoviridae (Circoviridae) genus Circovirus (Circovirus), no envelope, is the smallest animal virus found so far. According to the differences in pathogenicity, antigenicity and nucleic acid sequence, PCV can be divided into PCV-1 and PCV-2. PCV-1 is not known to be pathogenic; PCV-2 is pathogenic and can cause various clinical symptoms in pigs. The full length of PCV-1 genome is 1759bp, the full length of PCV-2 is 1768bp or 1767bp, and the sequence homology between the two is less than 80%. It has been confirmed that PVC has two main reading frames, ORF1 and ORF2. ORF1 is the largest reading frame and is involved in viral replication and transcription. ORF2 encodes the Cap ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/34C12N15/63A61K39/12A61P31/20G01N33/569C12R1/93
Inventor 张鹏超沈元嵇康孟超
Owner ZHEJIANG YEBIO BIOTECH
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