Detection Application Of N Gene Of Porcine Reproductive And Respiratory Syndrome Virus And Its Prokaryotic Expression Products

According to the published genomic sequence of PRRSV VR2332 strain ,apair of primers was designed and the nucleocapsid protein gene of VR2332 resp strain was amplified by RT-PCR.Then the amplified fragment and plasmid pET-32a were respectively digested by BamHI and Xho,the following lingaged reaction was operated. Finally the recombinant plasmid was constructed, designated pETN.pETN was transformed into the host cell BL21(DE3) and the expression procedure was optimized. Further study is under way to the development of new diagnostic method of PRRS.The recombinant nucleocapsid protein of PRRSV was obtained under the optimized condition of host cell cultivation and IPTG induction. Consequencely ,the expression product was purified by the means of His-bind resin protein purification procedure. Following SDS-PAGE and Westem-blot were used to detect the purification effect and the specificity of purified recombinant nucleocapsid protein of PRRSV. Then the purified N protein was used to be coated on the well of 96-well plate, each following step was optimized, such as coating concentration of recombinant nucleocapsid protein, sample diluent, chromogen(TMB), stop solution and its concentration. As a result ,an indirect ELISA was set up to detect antibody against PRRSV. About 200 serum samples were detected by the method and IDEXX ELISA kit, respectively. The agreement ratio between two method arrived at 91%.The purified recombinant nuclocapsid protein of PRRSV was used to conjoined with latex, the conjoined concentration and temperature were optimized. The method's sensitivity, specificity and stability were test. 50 serum samples were detected by the method and other two methods,N protein based ELISA and IDEXX ELISA kit. The result showed the recombinant N protein based LAT had certain agreement rate compared with otter two methods .The result indicates the LAT can be used to detect antibody against PRRSV.According to the nucleotide sequences of haemoagglutinin gene of Influenza virus and Porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 strain provided by GenBank, a pair of primers which includes main sequences of haemoagglutinin (HA) gene(33bp) was designed to amplify N gene (ORF7) of PRRSV by RT-PCR. The amplified fragment and pET-32a plasmid were digested by Bam HI and XhoI.Finally a recombinant plasmid named pETHN was constructed and was transformed into BL21(DE3). Consequently, the target protein was expressed by the method of IPTG induction ,and purified fusion protein was obtained by His-binding purification kit. As a result latex-agglutination test was set up and plenty of serum samples were tested by the method. The test result show the method has same sensitivity and specificity and has 81% accord rate compared with IDEXX ELISA diagnosis kit.