| Vesicular stomatitis is a disease of livestock caused by some members of the Vesiculovirus genus(Family Rhabdoviridae), two of which are called 'vesicular stomatitis virus'. Clinical disease presents as severe vesiculation and/or ulceration of the tongue, oral tissues, feet, and teats, and results in substantial loss of productivity. Except for its appearance in horses, it is clinically indistinguishable from foot-and-mouth disease. Using primers complementary to the conserved sequence of vesicular stomatitis virus (VSV) Indiana type genomic RNA previously published, we developed a RT-PCR method to detect vesicular stomatitis virus Indiana type. 5 7-day mice were challenged by hypodermic intradermic injection with VSV Indiana type, 5 3-week mice were challenged by brain injection, and then all the mice died within 2-5 days. Taking brains, liver, kidney, lymph nodes of the died mice. As the same time, tissue samples of 5 mice were taken from serology test. The 15 tissue samples and one from control mouse were tested, the results showed that all the ten samples from 7-day and 3-week mice challenged with VSV were positive and the sample from serology test and control mouse was negative. In addition, there was not nonspecific PCR product when foot-and-mouth virus (FMDV), swine vesicular disease virus (SVDV), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) were detected. All results suggested that the RT-PCR assay was specific fast exact for detection of the VSV Indiana type.The vesicular stomatitis virus Indiana type was propagated and harvested on BHK-21. One pair of unique primers were designed according to published sequence of VSV's cDNA. And the Nucleoprotein gene of vesicular stomatitis virus Indiana type was amplified by reverse transcription polymerase chain reaction (RT-PCR). The product of RT-PCR, which is about 1.3kb was cloned into pMD18-T vector, then the recombinant plasmid was digested by double restriction endonuclease EcoRl andHindlll to get N gene. The fragment was inserted into EcoRl and Hindlll multiple cloning site (MCS) of pET-32a expression vector. The recombinant of pET-32a-N was identified by PCR, corresponding restriction endonuclease and sequencing to be inserted into the open reading frame accurately. The recombinant vector pET-32a-N was ransformed into BL21(DE3), the expression capacity of the fusion protein were optimized by temperature concentration of BL21(DE3) before induced concentration of IPTG and the time of induced period. And the Nucleoprotein fusion protein was achieved a high expression level. Most of the recominant protein exits in sediment, and these proteins are inclusion bodies aggregated in BL21(DE3). After ultrasonicated, the sediment was isolated and purified. The expressed protein was valued by SDS-PAGE and Western blot. The results showed that molecular weight of the fusion protein accorded with the expected 65.3kDa. Using the recombinant and purified protein as antigen to coat micro-plate of 96 wells, an indirect ELISA assay was developed to detect anti-VSV sera. The elementary test shows that the recombinant protein is good as immunogen and antigen to be applied in ELISA.The recombinant plasmid pET-32a-N was cleavaged by double restriction endonuclease EcoRl and Notl to get N gene. The fragment was inserted into EcoRl and Notl multiple cloning site (MCS) of pPICZaA vector. The recombinant pPICZaA-N was identified by corresponding restriction endonuclease and PCR. The DNA linearized by Sacl was transformed into Pichia pastor is GS115 by electro and chemical poration. The expression vector pPICZaA-N was integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. As the same time, the Mut Phenotype of the recombinant Pichia strains identified by replica-plates procedure is Muts. Then the recombinant Pichia strains was induced with methanol. The expression products were tested by SDS-PAGE and Western blot. The results showed that the N gene had been expressed i... |
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