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Recombined rhabdovirus for expressing porcine bocavirus VP2 protein and application thereof

A technology of recombinant baculovirus and porcine boca virus, applied in the direction of antiviral agents, virus/phage, virus antigen components, etc., can solve the problems of stillbirth, death, respiratory tract infection, etc., and achieve easy access, high infection efficiency, high Target cell and tissue-specific effects

Active Publication Date: 2014-04-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of them (PBoV1, PBoV2 and PBoV4) were isolated from diseased pigs with PMWS, and the detection rate in diseased pigs was higher than that in healthy pigs, which indicated that porcine Boca virus had a certain correlation with the occurrence of post-weaning multisystemic wasting syndrome , it may also be mixed with pathogens such as porcine circovirus type 2, porcine blue ear disease virus and swine fever virus to cause more serious diseases, resulting in respiratory tract infection and diarrhea, as well as miscarriage, stillbirth, and even death

Method used

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  • Recombined rhabdovirus for expressing porcine bocavirus VP2 protein and application thereof
  • Recombined rhabdovirus for expressing porcine bocavirus VP2 protein and application thereof
  • Recombined rhabdovirus for expressing porcine bocavirus VP2 protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1: the preparation that contains the recombinant plasmid of porcine boca virus VP2 gene and recombinant baculovirus

[0068] 1.1 Construction of the baculovirus transfer plasmid pFastBac HTB-VP2 comprising the PBoV VP2 gene

[0069] Using the porcine Bocavirus genome isolated by our laboratory as a template, we designed a pair of specific primers for amplifying the VP2 gene according to the PBoV genome sequence reported by our laboratory (GenBank accession number is HQ223038.1), and introduced BamHI restriction site, XhoI restriction site was introduced into the downstream primer, and PBoV VP2 gene with specific restriction site was amplified by PCR. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd. The sequence is as follows:

[0070] VP2-F:5'-TTT GGATCC ATGTCCGCACACAGGGGGGC-3'

[0071] VP2-R:5'-GGG CTCGAG TTACAACACTTGGTTGAT-3'

[0072] The PCR product of VP2 gene was recovered, the PCR product was double-digested with restrictio...

Embodiment 2

[0100] Example 2: Identification of foreign proteins expressed by recombinant baculovirus

[0101] 2.1 IFA identification of recombinant baculovirus

[0102] The recombinant baculovirus rBV-VP2 was inoculated into a confluent monolayer of sf9 cells, and a wild-type baculovirus WTBV control was set at the same time. After the cells were observed to have lesions, the culture medium was discarded, fixed with pre-cooled 80% acetone for 5 min, and washed 3 times with PBS; blocked with PBS containing 10% goat serum (as the blocking solution and diluent of IFA) for 30 min; adding appropriate The diluted anti-His protein mouse monoclonal antibody is the primary antibody, act at 37°C for 1 hour, wash with PBS 3 times, 5 minutes each time; then add the secondary antibody (FITC-labeled goat anti-mouse IgG), and then wrap it in tin foil and act at 37°C for 45 minutes , washed 3 times with PBS, 10 minutes each time. The fluorescence of transfected cells was observed and photographed unde...

Embodiment 3

[0120] Embodiment 3: the preparation of recombinant subunit vaccine

[0121] Inoculate sf9 cells to 175cm 2 In the cell culture flask, when the cells grow to 70-80% confluent, infect the sf9 cells with the recombinant baculovirus rBV-VP2 of the P4 generation at a dose of MOI=5, culture at 27°C for 72 hours, collect the cell samples, wash with PBS for 1 The second time, cells were lysed by ultrasonication on ice for 20 min, and the lysate was centrifuged at 12,000 rpm at 4°C for 15 min to remove cell debris, and the supernatant was collected to purify His-tagged VP2 protein by nickel column affinity chromatography. A spectrophotometer is used to measure the concentration of the purified protein, and the supernatant containing the purified protein is mixed with an adjuvant to prepare a subunit vaccine.

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Abstract

The invention discloses a recombined rhabdovirus for expressing a porcine bocavirus VP2 protein and application thereof. A porcine bocavirus VP2 gene is amplified by PCR (polymerase chain reaction), and the nucleotide sequence of the porcine bocavirus VP2 gene is represented by SEQ ID NO.1. The amplified porcine bocavirus VP2 gene is inserted into BamHI and XhoI locuses of a rhabdovirus transmissible plasmid pFastBacTMHTB to construct a recombined transmissible plasmid pFastBac HTB-VP2; the recombined transmissible plasmid is converted into a DH10Bac competent cell comprising a rhabdovirus framework plasmid; the VP2 gene is integrated into the rhabdovirus framework plasmid Bacmid through homologous recombination; an sf9 cell is transfected by extracting the DNA of the recombined Bacmid to obtain the recombined rhabdovirus rBV-VP2 of which the preservation number is CCTCC V201401. A large quantity of VP2 protein and virus sample particles are expressed and produced in insect cells, and an effective PBoV VLPs subunit vaccine is developed.

Description

technical field [0001] The invention relates to the technical fields of animal virology and genetic engineering, in particular to a recombinant baculovirus expressing porcine boca virus VP2 protein and application thereof. Background technique [0002] Porcine Bocavirus (Porcine Bocavirus) was first found in Sweden in 2009 in sick pigs suffering from post-weaning multisystemic wasting syndrome (Post-weaning Multisystemic Wasting Syndrome, PMWS) and porcine circovirus type 2 co-infection (Blomstrom AL etc. Detection of a novel porcine boca-like virus in the background of porcine circovirus type2induced postweaning multisystemic wasting syndrome. Virus Res2009,146:125-129). In 2011, the nearly full-length genome sequence of porcine boca-like virus (PBo-likeV) was first reported in my country, and it was confirmed from phylogenetic analysis that PBo-likeV belongs to the genus Bocavirus, and it was named porcine bocavirus WH1 strain. Through epidemiological detection and analys...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/01A61K39/12A61P31/20C12R1/93
Inventor 肖少波方六荣刘丽枝马俊曾松林王荡罗锐陈焕春
Owner HUAZHONG AGRI UNIV
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