Prokaryotic Expression Of Gene RdRp,ORF3 And ORF5 Of PRRSV And The Establishment Of Indirect ELISA Methods

Porcine Reproductive and Respiratory Syndrome(PRRS), which was discovered in the late 1980's, is a highly infectious disease in the swine species, caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV). PRRS can lead to reproductive failures in the farrowing sow such as late-term abortions, an increased number of stillborn, mummified and weak-born pigs, and respiratory problems in pigs of all ages(especially among the youth).Infection of PRRSV can not only damage the immunity of the swine but also cause secondary affection or complicating disease involved other pathogenic microorganism, which exacerbates the situation. The disease is widely spread in many countries and regions in the world and has caused huge economic loss, which holds up the development of the world's swine industry. Since the outbreak of PRRS in 1995 in China, PRRSV has become one of the major pathogens that cause the swine's reproductive failures and respiratory problems in large scale swine industry.'High hot diease', a highly malgenic PRRSV,Which breaks out in the last two years, has been a sever impact toward the domestic swine industry. For more effective prevention the PRRS outbreak, this study focus on the clone and sequential analysis of the RdRp, ORF3 and ORF5 of PRRSV. The main content and result are as followed:1,According to the PRRSV's genetic sequence in GenBank, we design three pairs of special primers and clone PRRSV's RdRp, ORF3 and ORF5 genes into pMD18-T carrier by RT-PCR approch. And then we screen out the recombinant plasmid to sequence. As a result, we obtain gene in the size of 720bp, 585bp and 498bp.2,We clone gene RdRp, ORF3 and ORF5 to Prokaryotic expression vector Pet-32a(+), reconstructing plasmid Pet-RdRp, Pet-ORF3 and Pet-ORF5. Then they are conversed to BL21, induced by IPTG. Thus we obtain recombination protein which has around 46KDa, 42KDa and 38KDa.3,By using His-Bind Protein purification kit, we depurate a large amount of recombinant protein. Then we use these depurated Recombinant protein as Diagnostic antigen, and by discovering ELISA conditions such as concentration of antigen, concentration of serum and effective duration of serum, we sketchily establish a set of indirect ELISA methods to detect PRRSV antigen.This study successfully isolate and identify PRRSV's epidemic strain cause swine'high hot disease'in Guangdong. As well, it provides a elementary research result on clone of the genes of RdRp, ORF3 and ORF5 and Protein expression and indirect ELISA methods. This research further discloses PRRSV's Non-structural protein and Structural protein's molecule characteristic, provides theoretical foundations for PRRSV molecular epidemiology researches and offers innovative material and theoretical references for PRRSV's diagnose and prevention.