Expression And Purification Of N Gene Of Porcine Reproductive And Respiratory Syndrome Virus And Establishment Of N-ELISA

In this research, the N gene of PRRSV CH-1R Strain was amplified, then subcloned into the prokaryotic expression vectors, prokaryotic expression and the initial establishment of the ELISA detection methods.1. Cloning and bioinformatics analysis of the N geneBased on the sequence of N gene of PRRSV registered in GeneBank (SignNo. EU807840.1), we designed and synthesized a pair of specific primers, extracted the total RNA of the PRRSV CH-1R Strain as template, and amplified the N gene by RT-PCR. Then the gene fragment was cloned into pMD19 T-Simple Vector. We constructed the recombinant plasmid pMD-N. Enzyme digestion and sequencing revealed that the length of the sequence was 386 bp including a 372bp ORF which coded 123 amino acids and shared 92.7%-95.7%nucleotide sequence identity and 93.5%-96.0% amino acid sequence homology compared with American type virus strains. And the similarity of the nucleotide sequences and the amino acid sequences are respectively 65.3%-65.9%, and 57.3%-58.1% among Europe type virus strains.2. Prokaryote expression and activity detection of the N proteinThe pMD-N plasmid and the prokaryotic expression vector pET-32a (+) were digested by restriction enzymes EcoR I and Sal I to construct the prokaryotic expression plasmid pET-N. Then the recombination prokaryotic expression plasmid pET-N was transformed into E.coli Rosetta TM(DE3). Induced by IPTG, the recombinant protein was expressed. Its molecular weight was 34kD and the recombinant nucleoprotein was in form of the inclusion body as well as soluble by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The fution protein N showed specific reactogenicity by Western blot (immunoblotting experiments).3. Purification of N protein and establishment of ELISAAn indirect ELISA was developed based on purified PRRSV nucloprotein.Its optimal reaction conditions were:coating antigen at concentration of 13.3μg/mL,37℃1h and 4℃overnight,5% defatted milk powder blocking at 37℃2h, serum sample (1:40) at 37℃for 90min, HRP labeled Rabbit anti-Pig IgG (1:4000) at 37℃for 40min, the substrate TMB for ELISA being incubated at room temperature for 15min. The threshold of positive and negative serum was 0.434 by statistical analysis. The ELISA assay was repeatable, sensitive and specific. The sensitivity was 95.6% and specificity was 93.0% of the ELISA, comparing to the IDEXX kit, the coincidence rate was 94.7%.The result of experiments showed no significant difference between the two assays.On this experiment, the N gene of the PRRSV was cloned and expressed. An indirect ELISA is established based on the purified recombinant N protein. The bio-information, molecular biology, immunogenicity of N gene were studied. This research provides reference for more detailed study on PRRSV.