Establishment Of The Diagnostic Method For The RT-PCR And ELISA Of Vesicular Stomatitis

Vesicular Stomatitis Virus (VSV), which causes acute, febricity, height taction anthropo zoonosis, is a number of the family Rhabdoviridae, order Vesicu1ovirus. The clinical signs of Vesicular Stomatitis (VS) are characteristic of water vacuole in mucous membrane of mouth, tongue, lip, papillae, and coronet, which are similar with Foot-and-Mouth Disease (FMD) and Swine Vesicular Disease (SVD).VSV is classified into 2 serotypes on the basis of antigenicity: New Jersey (NJ) and Indiana (IN). VS is one of the main infections diseases harmed domestic animal. Since its breakout in Mexico in 1968, VS has occurred frequently in American, which caused serious economic loss in the cultivate industry. So it is important to establish a quick and accurate diagnosing method to prevent or control of VS.Glycoprotein (G protein), one important surface and protective antigen of the VSV, determine virus infectivity. Even if the nucleotide sequences of various genomes among the distinct strains of VSV are different, the variation of glycoprotein gene is biggest. In present study, on the basis of the gene of G protein of VSV, a RT-PCR method was established to detect VSV. The RT-PCR method is high sensitive and specific. Additionally, G protein of VSV was expressed and was used to envelope antigen to establish an ELISA method for the diagnosis of antibody against VSV.The design of primer for RT-PCR was based on the nucleotide sequence of G gene of the Indiana strain in Genbank. The predicted amplified product is 1346bp. The recovered target fragment was linked with pMD18-T vector, was transformed Ecoli.DH5αcompetent cell, was picked white bacterial colony to extract plasmid. The recombinant plasmid was sequenced after the PCR identification. The homology between target gene and other genes of VSV in Genbank was above 98.9%, which showed VSV G gene was obtained through amplification. In the present study, through the detection of specificity and sensibility, a RT-PCR method was established. The experimental sample was detected by the RT-PCR method, the result showed that the positive rate was 100%.G gene was subcloned into prokaryoutic high-expressing vector pET-28a(+). We used vector's general premier to sequence pET-28a(+)-G recombinant plasmid. The result showed that the sequenced G gene was identical with the protosequence and the target gene which was inserted into expressing vector had uniformity, and had correct direction and site. BL21 competent cell was transformed by positive recombinant plasmid and was induced to express by IPTG. Expession product being analysized through SDS-PAGE, target protein and vector multimetric histidine protein were confluently expressed, and a target approximative band of 57kDa. Then we studied the expressing condition of recombinant protein. There are more recombinant proteins expressing with more time and 5h after being induced, recombinant protein reach its maximum. The expressing quantity of recombinant protein is influenced by the concentration of IPTG In the condition of 1mmol/L IPTG being induced 5 hours, the extrinsic protein of bacteria is more than in any other conditions.In order to obtain active protein of nature conformation, we optimized the condition for extraction and purification of recombinant protein from cytorrhyctes of bacteria. In the present study, we obtained high purity recombinant protein through Ni2+ affinity chromatography. The purity of protein reached 80%-85% by renature; the purified G protein was analysized with VSV positive serum by Western Blotting. G protein expressed product is approximately 57kDa. The study offered necessary experiment material and basis for further study constitutive protein's constitution and function of VSV Indiana strain. It will help to develop specific diagnosing kit.In the present study, the fusion protein was used as envelope antigen to detect experimental positive/negative sera by ELISA. An indirect ELISA method for antibody against VSV was established though ascertaining the optimal envelop concentration of recombinant glycoprotein, the optimal dilution factor of sera, and the optimal action time and substrate visualization time of enzyme linked antibody. The ELISA method for detecting the antibody against VSV in serum was high sensitive and special.