Detection Of Swine Vesicular Disease Virus RNA And Expression Of VP1 Gene In Escherichia Coli

Swine vesicular disease is a highly contagious disease of pigs. Symptoms are clinically indistinguishable from those caused by foot and mouth disease virus and swine vesicular disease is therefore classified as a list A disease by the Office International des Epizooties(OIE). Since this disease was out break, detection and diagnosis of the swine vesicular disease and its molecule characteristics have been reaserched in and out of our country.In order to meet with export and import quarantination, further reaserch was finished about detection of the swine vesicular disease virus (SVDV) RNA and expression of VP1 gene in Escherichia coli, bases on many referenced articals were founded.Part1 Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction:Two polymerase chain reaction (PCR) assays are described for detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR).Both the RT-PCR and RT-nPCR were used on condition that two pair of primers were designed according to several reported complete nucleotide sequence of SVDV isolated in GenBank and other important vesicular disease viruses such as foot-and-mouth disease virus,vesicular stomatitis virus(VSV) .vesicular exanthema of swine vierus and so on. At the same time, this assay was determined its specificity and sensitivity. The results showed that this assay has perfect specificity, detecting SVDV were all positive, while those of FMDV and VESV were all negative; its sensitivity: the first amplification can detect 100TCID₅₀/mL,the second can detect 0.1TCID₅₀/mL. Analogue tissues were detected by the above assay. The results showed that it can detect analogue tissueseffectively.The results showed that the RT-PCR and RT-nPCR were useful for the diaganose of SVDV and epidemiologically investigate swine vesicular disease.Part 2 Analysis of VP1 coding nucleotide sequence of swine vesicular disease virus:RNA of an the SVDV strain was extracted by the single-step method of RNA isolation. About 872bp, including VP1 gene, was amplified by the reverse transcription nested PCR (RT-nPCR) with one pair of primer designed.After purified, the PCR products was ligased with pMD18-T vector, then the ligased products were transformed into Escherichia coli. Positive cloned Escherichia coli was selected and indentified .The nucleotide sequence of VP1 was determined by the dideoxy-mediiated chain termination method. The result showed that the nucleotide sequence of VPlwas obtained correctively. The homologies of nucleotide sequence and deduced amino acid sequence of VP1 were analysised compared to the VP1 region of these reported SVDV. Homology of the nucleotide sequence was between 89% 98%, and that of deduced amino acid sequence was between 89% 98%.By comprised with these difeferences,varianced amino acid were fined in the deduced amino acid sequence of VP1 gene of SVDV,according to the referenced amino acid sequence of VP1 gene of SVDV.These varianced sites were 7,131 and 136,and residued amino acids were Met,Val and Thr.Part3 Cloning of structural protein VP1 gene of swine vesicular disease virus and its expression in Escherichia coli:A pair of primer,containing Xho I and Nco I sites, was designed according to the result of DNA sequence.A DNA sequence .about 885bp,was amplified by PCR methodising recombinant plasmids as template.The PCR products amplified and expression vector pET-30 c(+) were both digested by Xho I and Nco I respectively.After purified, Thetarget gene VPlwas subcloned into this vector, and acquired the recombinant plasmids for expression. Then therecombinant plasmids were transformed into Escherichia coli BL21(DE3) for VP1 expression ,and the positive clone was named as pET-30c-VPl .The insert position,the site and the reading frame of the insertion were identified correctly by PCR amplified and restricition digestion of the recombinant plasmids .The result showed that the insert site was correct in the expression vector.The target gene was induced to express VP1 protein in E.coli with IPTG.The induced bacteria cultures were collected at certain different time and diposed with 2xSDS sample buffer. subsequently these samples were examined by SDS-PAGE and Western-blot analysis.The results indicated that the transformed BL21(DE3) by the recombinant plasmids could express the whole structural protein VPlof SVDV.The molecular weight of the fusion protein was about 37 kD, and it can be recognized by the positive serum of SVDV.