Expression And Purification Of N Gene Of PRRSV Isolate From Hebei And Establishment Of N-ELISA

Porcine reproductive and respiratory syndrome(PRRS) characterized by reproductive failure in sows and respiratory disease in piglets,is caused by porcine reproductive and respiratory syndrome virus(PRRSV),is also known as the Porcine Blue ear disease. PRRS has become a infectious disease of the mainly affects in the healthy development of the current China pig industry.In this study, a PRRSV was isolated from the local Hebei province and compared with the nucleotide sequence and amino acid sequence from Gen Bank, wich can grasp the PRRS variation trend of molecular epidemiology in Hebei. As the Highest levels and immunogenicity in PRRSV,the ORF7 gene was cloned into pet-32 a vector to generate the recombinant expressing plasmid,and an indirect ELISA for detection of the antibodies to PRRSV was developed using the N protein on the basics of the isolated virus.In the study a porcine productive and respiratory syndrome virus field strain, tentatively named HB-XL, was isolated from suspicious PRRSV-positive materials of sick pigs in Xinle areas, Hebei province according to the Marc-145 cell culture and RT-PCR detection method. Further analysis by cloning ORF5 and NSP2 sequencing, homology comparison and phylogenetic evolutionary tree showed that the ORF5 and NSP2 of HBXL share the similar mutant genetic relationship to the majority of the domestic epidemic PRRSV strains,and the relatively distant relationship to the American classical strains and recombinant strain QYYZ, and the Europe strain LV is an independent branch; The ORF5 gene was consist of 200 amino acids and the site of 13 and 151 were Argrinine(R) which were related to virulent strains, and the site of 137 was serine(S) indicating that the isolated strain was wild strain; The NSP2 gene had a discontinuous deletions of 3 bases and 87 bases compared with the American classical strains.The results showed that PRRSV HB-XL strain belongs to the American type PRRSV, and with a trend of continuous variation.A pair of specific primers was designed and synthesized according to the published N gene sequence of PRRSV from Gen Bank, and the N gene of PRRSV strain HB-XL was am plified by RT-PCR. The N gene was cloned into p ET-32 a vector to generate the recombina nt expressing plasmid p ET-32a-N. Then p ET-32 a was transformed into the host cell BL21(DE3) and the expression procedure was optimized. The fusion protein was successfully ob tained with the induction of 1.5 m M IPTG,37℃,4h. The fusion protein could specifica lly react to antiserum against PRRSV by Western blotting. An indirect ELISA for detection of the antibodies to PRRSV was developed using the purified nucleoprotein,and its optimal reaction conditions were determined: coating antigen at 37 ℃ 1 h or 4 ℃ overnight at a concentration of 2.23μg/ml,2% gelatin as blocking agent at 37 ℃ 1 h,serum sample(1:80)at 37 ℃ 1h,HRP-conjugated antibody(1:1000)at 37 ℃ 40 min, the substrate T MB for ELISA being incubated at room temperature for 15 min. The OD450>0.363 is posit ive and the OD450<0.34 is negative. And compared with the IDEXX Test kit,thesensitivity and specificity of the ELISA were 92.3% and 90.4%,respectively,whichshowed no signif icant difference between the two assays.The coincidencerate of the two assays was up to 91.6%.