The Cloning And Bioinformation Analysis Of ORF5 Gene Of PRRSV-SCQ Strain & Prokaryotic Expression Of DORF5 Gene

Glycoprotein 5 (GP₅) is one of the major structural proteins andimmunoprotection proteins of porcine reproductive and respiratory syndrome virus(PRRSV),it can induce the production of neutralization antibody,therefore,it is regarded as agood material for the designing of new type molecule diagnostic reagent and new generationvaccine.In this study,the total RNA of cells-PRRSV was extracted ,then the ORF5 gene of PRRSV-SCQ strain was amplified by RT-PCR and cloned to pMD18-T vector.The hydrophobicity, transmembrane helices, signal peptide, latent antigen site and secondary structure of PRRSV-SCQ strain GP5 protein were predicted by biological software and servers online.And then the relationship between its construction and function was discussed. GP5 contains three transmembrane regions,six main predicted epitope and the signal peptides is 1-31 amino acids residues,the C-terminal contains many random coils.Based on these predicts, a couple of primers was designed and the gene segment dORF5 gene deleting N-terminal signal peptide(also one of the transmembrane helices) was amplified from the recombinant plasmid pMD18-T-ORF5 by PCR. The segment dORF5 was cloned into expression vector PBAD/Myc-His B and the recombinant plasmid PBAD/Myc-His B-dORF5. was constructed successfully. The PBAD/Myc-His B-dORF5 was identified by PCR, restriction endonucleases digestion and by sequencing. The PBAD/Myc-His B-dORF5 was transformed into E.coli TOP10 cells and the cells were induced by L-arabrose.The SDS-PAGE test indicated that the GP5 protein was successfully expressed in E coli.cell. The molecular weight was about 19.4 kDa and it can amount to 17.1% of the total mass of bacterial proteins.The Western-blotting test showed that the protein had immunological reactive activity.This study provided fundamental data and materials for the further study on the structure and function of GP5 protein,as well as new type molecular diagnostic reagent.In addition,it also indicates that bioinformation is significant for designing experiment.