Cloning And Expression And Application Study Of N And M Gene Of High Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Acoording to the high pathogenic PRRSV nucleotide sequences published in Genbank, two pairs of primes for N and M genes were designed, respectively to amplify the corresponding target genes by RT-PCR. Then the amplified fragment and plasmid pET-28(a) and pBV220 were respectively digested by Ecol I and Sal I, the following ligation reaction was operated. Finally the recombinant plasmid was constructed, designated PET-28N and pBV220M. The recombinant plasmid of PET-28N and pBV220M were respectively transformed into the host cell BL21(DE3). Positive clones were screened and identified by PCR and enzyme-digestion. Except termination codon sequencing results showed N and M genes contained 372bp and 525bp nucleotides.The recombinant plasmid were transformed successfully into the host cell BL21(DE3), the expression procedure was optimized. The results of SDS-PAGE and West-blotting indicated that the N and M protein were expressed successfully. The results that the recombinant N and M protein molecule weight were about 17 kD and 22 kD respectively.Using the purified recombinant protein, an indirect ELISA for detection of anti-PRRSV antibodies was developed and its optimal reactions were determined: the optimal coationg concentration was 0.5μg/ml(N) and 1.0gμ/ml(M); the optimal dilution of serum examined was 1:40. The results showed that the indirect N-ELISA and M-ELISA methods had advantages of good repetition, high sensibility and strong specificity by repeated test and crossing test. Compared with the IDEXX test kit, the sensitivity, specificity and coincidence of the established methods were no significant difference by statistics analysis.