| HEV (Hepatitis E, HE), which can be a contagious zoonoses to animals and human beings caused by the hepatitis E virus. It is mainly spreaded from the feces to mouth, usually after the source of drinking water being contaminated; it will cause an outbreak of epidemic. There were sporadic cases globally. It had ever been occurred the world's largest epidemic of hepatitis E in Hetian, Kashi and Kezilesu Kerkezi Autonomous State, which in the south of Xinjiang of China, in the eighties of last century. In addition, more and more data show that, HEV not only spread in humans, is also widely distributed in a variety of animals. In a wide range of animals from all over the world exist the natural HEV infection, and HEV isolated from animal and human had very high similarity, while human HEV have been successfully infected on a variety of animals experimentally. Islam as a faith-based religion in mainly minorities living in the Xinjiang region, the sheep is not only the most important livestock, but also the livestock closest contacted with the production and with people's daily lives. So it not only has great significance to handle the epidemiological situation of sheep for human's public health, but also to explore the host range of viruses, with little incidence of infected animals, how to spread between species and so many unknown issues. HEV antibodies were detected with double antigen sandwich enzyme-linked immunosorbent assay (DS-ELISA) for clarifing the HEV infection distribution on sheep in the different regions of Xinjiang and recombination cloning vector HEVORF2-62 and pET-28a prokaryotic expression vector had successfully been constructed, so it established the foundation for early indirect ELISA diagnostic method to HE.In order to learn prevalence situation of sheep hepatitis E virus infection in Xinjiang, we start this experiment on HEV.1131 sheep sera from 14 regions of Xingjiang (357 from Southern Xinjiang, 774 from Northern Xinjiang) were tested with DS-ELISA for the presence of anti-HEV IgG, and the prevalence differences were analysed based on location(South and North Xinjiang),age range(≤lyear,2-3years,≥3years) and species of sheep by X2 test. The result showed the overall prevalence of anti-HEV was 5.22%(59/1131), and 4.76%(17/357) for the South Xinjiang,and had no significant differences (P>0.05) than that of the North 5.42%(42/774). The prevalence rate of Sheep in spring (1.53%(7/458)) was significantly higher (P<0.05) than that of the Sheep in the fall (7.73%(52/673)). The result showed the sheep of Xinjiang has HEV in generally.In this paper, pig source ORF2-fusion gene, and express this in pET-28a prokaryotic expression vector were recombinated. Two pairs of degenerate primers were designed according to the genomic sequence of HEV landed submitted on Gene bank, ORF-62 gene 187bp was cloned by RT-PCR technology, and recombinatedwhich would be cloned into vector pBS-T, thus positive clones were got, then after sequencing, ORF-62 fusion gene was successfully constructed, named T-ORF-62.The vector T-ORF-62 and pET-28a were excised by BamHI, Hindlll respectively.225bp fragment and 5.3Kb vector fragment Recycling, then using T4 DNA ligase connected this two fragments to construct a recombinant prokaryotic expression plasmid pET-ORF-62. Through digestion and sequencing, the correct reading frame and the positive recombinant plasmid was obtaine.This recombinant plasmid was transformed into DE3 host expressing bacteria by IPTG,which has four density (0.8mM,1.0mM,1.2mM,1.4mM), for inducing and expressing then for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Dot blot hybridization showed that expression of recombinant strains under the IPTG density of 1.2mM can successfully express ORF-62 fusion protein. |
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