Isolation And Prokaryotic Expression Of Neucleoprotein Of PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is an urgent contagion and highly infectious swine disease, which is caused by porcine reproductive and respiratory syndrome virus(PRRSV). The disease was characterized by fever, inappetence and respiratory disease in pigs of any age, abortion or increased proportions of stillbirn and infirm piglets in pregnant sows and high mortality in pre-weaning piglets. This viral disease was first discovered in the United States in 1987. Subsequently found in Europe, it spread rapidly and became endemic in most of the major swine- producing areas throughout the world, causing great economic losses worldwide each year. Unparalleled large-scale outbreaks of an originally unknown, but so-called"high fever"disease in China in 2006, it had confirmed the essence is the variant of PRRS. PRRSV belongs to the family Arteriviridae within the order Nidovirales. According to the different virus genome, the isolates had been divided into the Northern American and the European strain, the former is mostly epidemic in America and Asia, and"high fever"belongs to American strain after identification.A local strain of PRRSV was isolated from lungs of piglets in ChangChun swine farms, and named CC strain. The isolate could be propogated in MARC-145 cell, and cause typical cytopathogenic effect (CPE) was observed on cells, after three blind passages, but not do so in other cell lines, such as Vero,BHK-21,PK-15,F81 and DK cells. The virus was identificated by a series of systematic identification such as morphology test, RT-PCR, physicochemically test and serological neutralization test. The result showed that it was porine reproductive and respiratory syndrome virus(PRRSV).In this study, according to the published sequence of PRRSV genome, (e.g: VR-2332, GD and so on), five pairs of primers were designed, these primers are designed by molecular biology. The fragments of CC strain's Nsp2,ORF5~7 gene were obtained by RT-PCR, respectively. The products of PCR are purified and cloned into pMD18-T vector, and then transferred, the recombinant plasmid is identified by restriction endonuclease digestion then sent to be sequenced. Then the sequences of Nsp2,ORF5~7 genes of CC strain were compared with the sequences and deducted amino acid sequence of other domestic and foreign PRRSV strains has published in GeneBank by the biology software such as DNAStar. The results shown that the homology of deduced amino acid sequences of nonstructural protein 2 (Nsp2) gene among CC and other typical European strain LV was only 47%, and highly homologous to the new strain of China was above 98%. We observed these viral isolates, namely a discontinuous deletion of 30 amino acids(482aa, 534~562aa) in Nsp2. A novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, And it was higher than the homology between CC and typical American strains VR-2332, which was only 72%. The homology of deduced amino acid sequences of E, M, N gene among CC and other American strains was above 90%, 96%, 95%. Furthermore, it was found that the homology was very high among new strains of China(e.g: GD,HEB1,HuN,HUB,jiangxi-3 strains et.al), but the homology was very low among CC and LV. And the phylogenetic trees revealed that CC strain relationship are near with American type PRRSV, especially with several China strains, their relationship are quite near, may infer the isolate is height varivant. And it presumed that this isolate strain induced"high fever"in south and northeastward.Although PRRSV isolates identified from around the world cause similar disease in pigs, increasing data indicate that PRRSV strains are antigenically and genetically heterogenous and differ in virus in infected pigs. It is evident that the current vaccines are not effective in protecting against infections with the genetically diverse field strains of PRRSV, and the attenuated vaccine viruses can revert genetically to cause clinical disease. To establish a fast and effective method towards the disease is the important base for internal swinery`s investigate of epidemiology and immunity. This work will be a good basis to new generation vaccine, the diagnostic antigen and prevention of PRRS.N enclded from ORF7, is one of the main structure protein, N has highly conservative in serotype. It can detect antibody of PRRS, after infection PRRSV seven days, and can hold a month. It often takes detecting antigen.This study`s object is N of PRRSV. N gene fragment was amplified from the CC strain of PRRSV RNA by RT-PCR. And then, the fragment was cloned into pMD 18-T vector, obtaining a recombinant plasmid pT-N, after identification by restriction enzyme digestion and sequencing. N gene from the positive recombinant plasmid pT-N was inserted into the prokaryotic expression vector pET-28a(+). A recombinant plasmid pET-28a-N was constructed and identificated by restriction endonuclease digestion, then transformed into the E.coli BL21(DE3) plysS. The recombinant bacteria were induced with IPTG and the expression protein was analyzed by SDS-PAGE. The result showed that bacteria containing the positive plasmid induced it to express with 1mM IPTG and 4 hours. And a majority of the recombinant protein was expressed as inclusion bodies in E.coli. A unique band was detected with the molecular mass of approximately 18kDa. By analysis of Western blot, the expressed production was reactive with the positive swine serum of PRRSV, through gel thin layer scanning analysis, the amount of target protein is over 20% of the total bacteria protein. This showed that the high efficient expression of N was obtained which system of prokaryotic and eukaryotic expression. Using target protein after purified as coating antigen,an indirect ELISA is developed for detecting the anti-N antibody in the PRRSV serum by exploring the concentration of coating antigenand dilution degree of serum.On the whole, the study isolated and identified a new strain of PRRSV, and the indirect ELISA to detect PRRSV was established, which will provide a good fundament for development of PRRSV antibody surveillance kit.