Establishment Of Detection And Prokaryotic Expression Of ORF140Genes On Koi Herpesvirus

koi herpesvirus which is a virus that causes lethal disease in common carp and koi infection.Although the disease is highly contagious, other non-cyprinid species of the tilapia, grass fish,gold fish, gold fish and common carp crossbred, johnny carp and common carp crossbred that weren’t caused lethal. The disease with characteristics by pathogenesy time is short, a high mortality rate, outbreak of extensive area, rapidly propagation and so on. The virus induces the disease that often appears in every spring and autumn, when water temperatures is from16to25℃, and the most suitable temperatures is28℃. This disease has been outbreak and observed in many country in the worldwide, with a mortality rate from80%to95%. When koi herpesvirus outbreak on farm seriously,the farm’s mortality rate is as high as about100%, so it causing severe financial losses in fishery.At present,it has been publicly consider that the disease is the important infectious disease of caused death with common carp by fishery farmers and koi fans.koi herpesvirus is a member of the family Herpesviridae, which is a nenveloped virus containing, linear, double-stranded DNA genome packaged within an icosahedral capsid. It has156Open reading frames,8of which are duplicated in the terminal repeat, and contains an asymmetrical electron-dense region within the viral core. The main genome of viral containing thymidine kinase gene, polymerase gene, major capsid protein gene and triplex gene et al. ORF140gene encodes thymidine kinase gene,which is a virulence gene that play important roles in virus adsorption, penetration and replication in cells and exocytosis. koi herpesvirus has detective methods by neutralization tests, isolation and identification, polymerase chain reaction and fluorescent PCR,but these methods have many shortage,for example time consuming,high charge and complicated equipment which are not be used in large areas of clinical detection.Therefore,the establishment of a rapid,sensitive,special methods has important significant.First, Purification of virus from culture medium by three methods,one hand is extracted DNA and establishment a method of multiplicitas PCR were special to the TK gene and SPH gene. Another hand,the virus used as immunogen which is preparated and purificated polyclonal antibody agaist KHV. The titer of KHV antibody serum dilution were accomplished1:25600,and determine the useful concentration of antigen-antibody were1:1250,1:6400, and the final titer of purificated antibody serum were1:3200. Two methods were applied clinic detection which detected artificial infection koi examples and positive samples,fragement of155bp and570bp of TK gene sequence by double PCR,and the result shown that indirect ELISA method and double PCR were negative of control group. A pair of primers that were specific to the KHV TK gene were designed,a fragement of696bp of TK gene was amplified by PCR and was successful expressed in Escherichia coli.The expressive product were identifed by PCR and restriction enzyme Sph Ⅰ and EcoR Ⅰ. The result showed successful constructes prokaryotic expression system of KHV ORF140genes.SDS-PAGE analysis showed that the recombinant protein was38KDa band.The recombinant protein could react with KHV positive serum antibody by Western blot tests.The result showed that the recombinant protein shared a good specificity. The expression protein centrifugated and harvested supernatant liquid and precipitation,SDS-PAGE analysis showed that without expression protein in supernatant liquid,however and more expression protein in precipitation. The result declared that the expression protein are existence by inclusion body.