Cloning And Prokaryocyte Expression Of Nucleocapsid Gene Of Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) was an urgent contagion produced by porcine reproductive and respiratory syndrome virus (PRRSV), it has been broken in American in 1987 at the first time, subsequently spreaded in all over the world. PRRSV pathogen in homeland was first separated by Guo Baoqing, which reported in 1996. Now, this disease has become one of the major illness harmed swine breed aquatics. To establish a fast and effective method towards the disease is the important base for internal swinery's investigate of epidemiology and immunity.Nucleocapsid (N) protein was the major structural protein of virus, which encoded by ORF7.It has perfect immunogenic and highly conservitive in serotype. According to the PRRSV's sequence of American-type, one pair of primer included restricts enzyme cleavage sites HindIII and BamHI were designed that can amplify N gene. By RT-PCR, a gene fragment of about 372bp was amplified. And then, the fragment was cloned into pMD18-T vector. After the sequence determination was got, it was compared with sequence of American-type virus using blast tool and discovered that this gene was same to the American-type virus. Expressed plasmid pREST-N was constructed after N gene subcloned into pRESTB vector. The recombinant plasmid was transformed into E.coli BL21(DE3)PlysS, and induced with IPTG for expression. Then, the fusion protein about 17.7 ku was expressed. The size was accord with anticipate. By analysis of Western-Blot, the expressed production can react with antibody to PRRSV. The result indicated that nucleocapsid protein of PRRSV was expressed in the prokaryote system.Candidate: Li KaihangMajor: Preventive Veterinary ScienceSupervisor: Associate Prof. Jia Yongqing...