Cloning And Expression Of Gp5 Gene Jl/07/sw Strain Of Porcine Reproductive And Respiratory Syndrome Virus And Establishment Of Indirect Elisa Diagnosis Method

According to the gene order of PRRSV America type strains VR-2332 specific,one pair of primer was designed in order to amplify PRRSV JL/07/SW strain GP5 gene. The GP5 gene of PRRSV JL/07/SW strain have been successfully amplified by RT-PCR form the cell cultures of PRRSV J1/07/SW. to make use of pMD 18-T as vector to clone the GP5 gene. Sequencing showed that GP5 gene was 603bp at total length, encoding 201amino acids. Sequence analysis showed that the nucleotide homology between GP5 gene&VR-2332 strain and GP5 & LV strain was 99.0% and 67.8%, respectively. The Encoding amino acids homology was 99% and 63%, respectively.The GP5 gene was subcloned into pCI-neo expression vector, constructing the expression plasmid pCI-GP5. It transformed recombinant plasmid into the cells of Macr-145 and dentify expressed product by indirect IFA,SDS-PAGE and western-blot.The results showed that it had about 25KDa fusion protein, the protein were consistent with the expected. The results of the Western-blot immunoblotting assay demonstrated that the recombination protein possessed antigenic activity and could be specifically recognized by antiserum against PRRSV.Using recombined fusion protein as coating antigen,after analydid of western-blot,this recombination protein can make reflect with masculine blood serun of PRRSV,and used this purification of recombination protein set up a method of indirect enzyme-linked immunosorbent assay (ELISA) to diagnose PRRSV. The experiment made sure of the optimal primordial covering concentration was 2.30μg/mL. The optimal dilution of serum examined was 1:100. To check out anther disease of swine. positive serum, which weren't have no cross reaction by indirect ELISA method, it improved that this method had strong specificity.In this thesis, we studied the cloning and analysis GP5 gene of PRRSV. Applying to molecular biology technique to make GP5 gene of PRRSV with eukaryon expression vector'connection and establish indirect ELISA methods. At the same time, it made an intensive study of the feature of etiological agent molecular biology for PRRSV JL/07/SW strain, also established a preliminary diagnostic method. This research had a significant importance in epidemiological investigation, etiological study, and diagnosis and control of PRRS,which foundation of study on funtion of structural gene of PRRSV JL/07/sw.