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African swine fever P30 protein recombinant baculovirus expression vector and preparation method thereof

A technology of recombinant baculovirus and African swine fever, applied in the direction of virus/bacteriophage, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of complex infection and escape immune mechanism

Active Publication Date: 2016-06-08
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ASFV storage hosts and vectors are soft ticks and African wild boars. The mechanism of infection and escape immunity of the disease is very complicated. At present, there is no vaccine that can prevent and control it. In order to avoid the sudden outbreak of African swine fever in my country, it is necessary to establish a rapid and accurate method Methods of detecting and diagnosing the disease

Method used

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  • African swine fever P30 protein recombinant baculovirus expression vector and preparation method thereof
  • African swine fever P30 protein recombinant baculovirus expression vector and preparation method thereof
  • African swine fever P30 protein recombinant baculovirus expression vector and preparation method thereof

Examples

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Embodiment 1

[0028] Example 1: Construction of recombinant baculovirus vector containing ASFVP30 gene and acquisition of recombinant shuttle bacmid rBacmid-ASFV-P30.

[0029] 1.1 Primers were designed according to the CP204L gene in the ASFV genome sequence published by GenBank (accession number: FN557520.1), and the PCR product of the ASFVP30 gene was amplified from the cloning plasmid PCR-4TOPO-P30 of the African swine fever virus P30 gene. The sequence after the upstream primer p30F is added to the BamH1 restriction site is: 5'-CGG GATCCA TGCACCATCATCATCATCATATGGATTTTATTTTAAATATATC-3', the sequence after the downstream primer p30R is added to the Xho1 restriction site is: 5'-CCG CTCGAG TTAGTGGTGGTGGTGGTGGTGTTTTTTTTTTAAAAGTTTAATG-3', the underlined part is the restriction site, and 6 histidine tags are added to both ends of the protein sequence. Using the PCR-4TOPO-P30 recombinant plasmid as a template, the full-length P30 gene with His at both ends was amplified, which was about 639b...

Embodiment 2

[0033] Example 2: Expression of African swine fever P30 protein in insect cells.

[0034] 2.1 Transfection of insect cells

[0035] (1) Take 2ml of well-grown Sf9 insect cells and count about 2×10 6 Add cells to a six-well plate and allow cells to attach for 3 hours;

[0036] (2) Prepare two 1.5ml sterilized centrifuge tubes and mark them well. Add 5 μL of Bacmid-ASFV-P30 and 100 μL of Grace medium without antibiotics and FBS to one of the centrifuge tubes, and mix gently. In another centrifuge tube, 6 μL of CellfectinReagent and 100 μl of Grace medium without antibiotics and FBS were mixed. Cellfectin Reagent is fat-soluble, and it will precipitate if left for a long time. It is best to shake it evenly before use.

[0037] (3) The solutions in the two tubes were mixed and incubated at room temperature for 45 minutes.

[0038] (4) Add 800 μL of Grace medium. Then the Grace medium in the six-well plate was removed, washed once gently with Grace medium (without antibiotics ...

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Abstract

The invention provides African swine fever P30 protein recombinant baculovirus expression vector and a preparation method thereof. The method comprises: amplifying in plasmid PCR-4TOPO-P30 of ASFV (African swine fever virus) P30 full-length gene to obtain P39 gene, linking the amplified P30 gene to a baculovirus vector pFastBac 1 to construct recombinant baculovirus vector pFastBac1-ASFV-P30, converting into competent Escherichia coli cells DH10Bac to obtain recombinant shuttle bacmid rBacmid-ASFV-P30, transfecting to insect cells Sf9 after verification is correct to obtain recombinant baculovirus, and passage amplifying the recombinant baculovirus, linking baculovirus high in titer and containing ASFV P30 gene to High Five insect cells for eukaryotic expression of ASFV P30. The African swine fever P30 protein recombinant baculovirus expression vector is constructed by using the method, a recombinant baculovirus expression system is used to express African swine fever P30 protein in insect cells, and basis is laid for African swine fever ELISA (enzyme-linked-immunosorbent serologic assay) detections.

Description

technical field [0001] The invention relates to the field of gene recombination expression in biotechnology, in particular to an African swine fever P30 protein recombinant baculovirus expression vector and a preparation method thereof. Background technique [0002] African swine fever (African swine fever, ASF) is an acute, highly contagious and highly lethal infectious disease of pigs, caused by African swine fever virus (Africans swine fever viruses. . The disease was first discovered in Kenya, and later the epidemic spread in Africa, Europe and other countries. In recent years, the epidemic of the disease has expanded and spread to neighboring countries such as Georgia and Russia. In China, the development of the pig industry is facing a huge threat. ASFV storage hosts and media are soft ticks and African wild boars. The mechanism of infection and immune escape of the disease is very complicated. There is no vaccine that can prevent and control it. In order to avoid th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66
CPCC12N15/86C12N2710/14043
Inventor 单虎王玉英于泽坤秦志华张洪亮黄娟
Owner QINGDAO AGRI UNIV
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