Cloning Of M And N Genes Of Porcine Reproductive And Respiratory Yndrome Virus (PRRSV) And The Expression And Application Of N Gene

Porcine reproductive and respiratory syndrome virus (PRRSV) belonging to the Arteriviridae family,which causes reproductive disorders in gilts and sows and respiratory distress in pigs,is an economically important etiological agent of modern porcine industry. Previous studies have demonstrated that M and N proteins contain the conseved epitopes among PRRSV isolates.Especially, N protein is the most abundant protein with high antigenic activity of PRRSV, therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease caused by PRRSV. In this study, complementary DNA corresponding to the entire sequence of M and N gene of the C14-2 strain of PRRSV were cloned into vector pMD18-T after being amplified by reverse transcription polymerase chain reaction (RT-PCR) method. Then N gene was subcloned into prokaryotic vector pET-32a(+) to express recombinant N protein for the purpose of developing an indirect rN-ELISA method to detect antiserum against PRRSV.According to the nucleotide sequence of PRRSV VR-2332. two pairs of primes P1\P2 and P3/P4 for M and N genes were designed ,respectively, to amplify the corresponding target genes byRT-PCR. After purified, the gene fragments were both cloned into a vector pMD18-T. Positive clones were screened and identified by PCR and enzyme-digestion. Except termination codon, sequencing results showed M and N genes of isolate C14-2 contained 522bp and 369bp nucleotides, correspondingly encoding 174 and 123 amino acids, respectively. The M and N genes and the deduced amino acids sequence were compared with corresponding sequence of VR2332 and LV. The results indicated that the homology of M gene and the deduced amino acids were 99.0%, 67.8%, 99.7% and 65.1%, respectively. The homology of N gene and the deduced amino acids were 98.3%, 77.5%, 99.2% and 57.7%, respectively. The phylogenetic relationships which confirmed that the isolate C14-2 blonged to American genotype strains, were established using neighbour-joining and parsimony methods based on M and N gene nucleotides.The N gene contained in pMD18T-N were subloned into prokaryotic vector pET-32a(+) to construct a recombinant plasmid pET-32a-N. After recombinant plasmid...