Cloning And Sequencing Of Prrsv And Preparation Of Monoclonal Antibody And Cloning And Expression Of Sialoadhesin

Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly caused reproductive disorder, including of premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages and high mortality. The disease was first discovered in American in 1987 and then prevailed in many countries.In 1991, this disease was discovered in Taiwan.In 1996, Guo Baoqing first isolated a PRRSV strain from aborted fetuses and verified the evidence of PRRSV in our country. In 2006, there was large outbreak in our South districts and this disease was diagnosed as highly pathogenic PRRS.The disease caused high morbidity and mortality and the clinical symptom became more complicated and the prevalence also was gradually expanded.At the present, PRRS has prevailed in the main worldwhile pig-raising country and districts and caused pig-raising large economic losses in the world.This subject can be divided into three parts:1 Isolation and sequence analysis of PRRSV SD-TA mutational strain.In 2008, one PRRSV(SD-TA strain)was isolated from the'Ardent Fever Disease'in pigs which could react with anti-PRRSV positive sera. The ten pairs of primers were designed respectively as the gene sequences of PRRSV classical strain (VR-2332) and mutational strain (JXA1) from GenBank and the aimed gene fragments were amplified respectively by RT-PCR. Then amplified cDNA fragments were cloned into the vector pMD18-T and sequenced. The sequences were analyzed compared with the sequences from the strains in and aboard the country by the software DNASTAR. The results showed that the nucleotides homologies were 89.3%～99.1% between PRRSV SD-TA strain and American strain and 30 nucleotides in the fragments of Nsp2 were deleted. Compared with European strain LV, the nucleotides homologies were 59.9%.The genetic relationships showed that SD-TA strain was the highly pathogenic PRRSV strain.2 Expression of PRRSV-N Protein and preparation and characterization of monoclonal antibodies.The recombinant plasmid pET-30a (+)-ORF7 of N protein of PRRSV VR-2332 strain was identified and then transformed into Rosetta competent cell. The N proteins were expressed by the induction. The expressed recombinant proteins were 21KD by SDS-PAGE analysis. The Balb/c mice were inoculated with the purified fusion proteins and then the splenocyte were fused with myeloma cell SP2/0. The supernatants of the fusion cells were measured on the coated antigens of the purified fusion proteins by ELISA. Then, one hybridoma cell against N proteins were stably obtained by limited dilution assays and named as 6D10. By Western-blot analysis, the Mab 6D10 could react with the N proteins specifically. And, the Mab 6D10 could react with classical strain (VR, BJ and MLV vaccine strain) and highly pathogenic mutational strain (SD-TA, 2# and 4# strain) specifically.3 Cloning, sequencing, expression and purification of sialoadhesinThe 5 pairs of primers were designed based on the gene sequence of NM-214346 to amplify the gene sequence of Sn by RT-PCR.The prospective fragments were obtained and the size were as follows: 1125bp, 1062bp, 1101bp, 1182bp and 1200bp. The cDNA fragments were cloned into pMD18-T and sequenced.The recombinant plasmid pET-28a-Sn-1~5 were identified by restriction enzyme (EcoRⅠand SalⅠ) digestion and cloned into Rosetta competent cells and were induced and expressed.The sizes of expressed recombinant proteins were as follows: 41.4, 36.7, 38.5, 42.3 and 42.9KD by SD-PAGE as like as the prospective results. The obtained recombinant proteins reacted with anti-His MAb specifically by Western blot.