Swine Vesicular Virus Vp1 Gene Cloning, Expression And Detection Techniques Of Research

Swine vesicular disease (SVD) is also named swine infectious vesicular disease, caused by swine vesicular disease virus (SVDV). It is the causative agent of a highly contagious disease in pigs that causes vesicular lesions in the mouth and on the feet. It is difficult to distinguish SVD and foot-and-mouth disease virus (FMDV), because their clinical symptoms are familar. It infected our country animal husbandry and the international animal and its the product trade development, causes the serious public health question, the international veterinarian bureau (OIE) lists as A kind of infectious disease. In the international trade, most countries adopt the strict quarantine and the control measure to SVD have become one kind of technical trade protection barrier. Differential detection of SVDV from other vesicular disease virus of FMDV, Vesicular stomatitis virus (VSV) is important. As the vesicular lesions produced by these viruses are indistinguishable in pigs. Sensitive specific rapid safety and reliable methods to diagnosis the disease is essential for the implementation of control measures to prevent the spread of the disease.In this study, the biotechnologies such as gene clone and gene expression were applicated. VP1 coded the gene of swine vesicular disease virus outer covering protein antigen was cloned. The nucleus vector was constructed and the target protein was expressed. Some methods including enzyme linked immunosorbent assay (ELISA), RT-PCR, mutiplex PCR, Fluorescent real-time quantitative RT-PCR (TaqMan RT-PCR) for the detection of SVDV in clinical samples was developed.1. The VP1 gene of SVDV was amplified from RNA by the reverse transcription-polymerase chain reaction (RT-PCR), the product of which was a 849bp DNA fragment. Using T-A cloning technique, the PCR product was cloned into pMD18-T vector. The purified VP1 gene was re-cloned into pBAD/Thio TOPO vector. The recombinant plasmid was identified by PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP1 gene. The results of SDS-PAGE and Western blotting revealed that the VP1 protein was expressed in Escherichia coli TOPO10 in a high level and as recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. The optimal amount of the expressed fusion protein is 16%of total bacterial protein after being induced with L-arabinose at 0.002% concentration for 5 hours. It had a molecular mass of approximately 47.13 kDa and serologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of swine vesicular disease. An indirect enzyme-linked immunosorbent assay for swine vesicular disease using recombination antigen VP1 was established.2. According to released SVDV gene sequences and RT-PCR assay for the diagnosis and characterization of SVDV reported. Based on the conserver sequence of SVDV, 2 sets of primers to detect SVDV were designed. The product of the RT-PCR amplification is 251 bp and 366bp, subsequently cloned into pMD18-T Vector. The nucleotide of production was compared with the corresponding sequences of published sequence of SVDV. The nucleotide homology was 100%. Suggest that it is gene of SVDV. Specificity was tested by that amplification of FMDV VSV BHK21 cell was negative. The dilution of 10^-4 SVDV RNA can be detected. Suggest that it has high sensitivity. The RT-PCR method is rapid, sensitive and specific for detecting SVDV.3. The method of multiplex polymerase chain reaction was developed and optimized to distingwish FMDV SVDV and VSV. Three pairs of primers were designed base on the specific sequences of FMDV SVDV and VSV in GenBank. Samples containing FMDV SVDV and VSV genome were amplified desirablly by the multiplex PCR using the three sets of primers and then the multiplex PCR conditions were optimized. Three expected specific bands of FMDV gene 189bp SVDV gene 125 bp and VSV gene 300 bp in length. The multiplex PCR method proved to be rapid, sensitive and specific for detecting FMDV SVDV and VSV.4. A fluorogenic TaqMan RT-PCR assay using a primer/probe set designed from the VP1 region of the virus genome was developed for the specification and sensitivity detection of swine vesicular disease virus. The number of genome equivalents which were detect -ed 1 ×10² copies per reactions. No cross-reactivity was demonstrated when this primer/ probe sets were tested with RNA prepared from FMDV and VSV.the relationship between the values of cycle threshold (Ct) and the copy number Was linear(r=0.993). The fluorogenic RT-PCR provides relatively fast results, greater sensitivity, enables a quantitative assessment to be made of virus amounts and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure. The results suggest that it is seen as a valuable tool to complement the routine diagnostic procedures for SVDV diagnosis.In summary, SVDV VP1 gene was successfully expressed. The ELISA experiment was established. Some methods of RT-PCR, multiple PCR, and the real-time fluorescence quantitative RT-PCR to detect SVD was established. These methods for the prevention and the control of SVD , the export and import quarantine as well as the social public security regarding the SVD appears very important. And it has provided the technical strut to the diagnosis and the animal quarantine of SVDV for our country.