Screening Of Responsive Genes In Ducklings During Duck Hepatitis Virus Ⅰ Infection

Duck viral hepatitis is an acute,highly fatal and rapidly spreading viral disease of ducklings,which is caused by duck hepatitis virusâ… .The disease appear a brief pathognomonic syndrome of hepatitis.It is already distributed worldwide and is one of the most economic importance to all duck-growing farms because of the high potential mortality.At present,the research of DVH is mainly focus on sequencing the genome of DHV and methods for controling of the disease and so on.There have been,however,comparatively few research on the pathogenic mechanism of DVH. The research about the responsive mechanism of the host in the course of virus infection,the molecular mechanism of the rapid death of ducklings,the interaction of the proteins from host and virus is still a blank.To research the pathogenic mechanism of DVH,the research is involved in the differential expression of genes in the ducklings during DHV infection.The content of the research is as follows:1 Screening and Identification of responsive genes in ducklings during DHV infectionAnimal model is established by duck hepatitis virusâ… infect ducklings.By using Suppression Subtractive Hybridization technology,two subtractive cDNA libraries of the four mixed tissues composed of liver,spleen,kidney,brain of DHV infected ducklings and uninfected control are constructed.Out of the two libraries,fifty positive clones confirmed by PCR were selected for sequencing.After that,ten differential genes are obtained.These genes include six up-regulated genes such as matrix Gla protein(MGP),Lysozyme G,lymphocyte antigen 6 complex,locus E (LY6E),mucin 4,beta-2 microglobulin and a novel sequence;four down-regulated genes,which are IFN stimulated gene 12(ISG12),cytochrome c oxidase subunitâ…¢,leukocyte cell derived chemotaxin 1(LECT1),chromosome 14 open reading frame 100.The function of them can be divided into several aspects,for example,signal transduction,antiviral immunity,electron transport,and so on.To further confirm differential expression of seven interested genes in ducklings during DHV infection, the transcript abundance of them is assessed using semi-quantiative RT-PCR.The seven genes include MGP,Lysozyme G,LY6E,ISG12,cytochrome c oxidase subunitâ…¢,LECT1,chromosome 14 open reading frame 100.The result shows the seven genes are differential expression in ducklings during DHV infection. 2.Prokaryotic expression of ISG12 gene and function analysis by BioinformaticsAccording to the sequence of ISG12 gene in the reverse subtractive cDNA library, primers with restriction sites were designed and synthesized.The expectant fragments were amplified from liver tissue of duck embryos by common PCR.The fragments of ISG12 were subcloned into prokaryotic expressing vector pGEX-KG and expressed induced by IPTG in E.coli BL21.Analyzed by SDS-PAGE,ISG12 fusion protein present with about 35kD protein.The sequences of ISG12 protein have been analysised and predictied by biological software,we found ISG12 protein is localized in the nuclear envelope of the cell and have stronge antigenic.