Cloning Of DHV-Ⅰ VP1 Gene And Biology Characteristic Forecasts Of Structure Protein VP1

Duck Hepatitis typeⅠis one kind of acute highly lethal infectious disease of young duck which causes by duck hepatitis virus-Ⅰ.This virus mainly infect the 4-week-old ducks,especially less than seven days old ducks.The mortality rate may reach over 90%.So it is one of the most serious infectious diseases which threaten ducks.It was found in the United States in 1945,the cases of the disease in our Country was first reported in ShangHai.so far,there have different levels of occurrence and spread in duck feeding areas in our Country。Therefore,it has important significance to studying disease and etiological factors of the disease.We studied on the Virus RNA detection,the Cloning and analysis of the VP1,biological characteristics forecasts of duck hepatitis virus-Ⅰvaccine strain preserved in the laboratory.1.Accoding to duck hepatitis virus-Ⅰgenome sequence in Genebank,we designed one pair of primers in a relatively conservative region,used vaccine strain's RNA to carry out RT-PCR and obtained the expected size fragments.The results showed that the RT-PCR method had high sensitivity and specificity.The minimum detection of RNA template was 20pg.This method can only detect duck hepatitis virus-Ⅰ,and can not detect NDV,IBDV and normal duck embryo allantoic fluid.So,the estabished method of RT-PCR was specific,fast and sensitive,and can be used in the rapid Identification Of duck hepatitis virus-Ⅰ.2.Accoding to DHV-ⅠVP1 genome sequence in Genebank,we designed one pair of specific primers,extracted the total RNA from the allantoic fluid of infected chicken embryo.Used the RNA as template,we obtained 714bp specific band by RT-PCR to amplification VP1 gene.PCR products and plasmid PET32a(+)were cut by EcoRⅠand HindⅢ.recombinant plasmid PET/VP1 was constructed successfully and transformed into E.coli DH5α.The positive clone was sieved by PCR identification and enzyme digestion from transforman(?),and identified by agarose gel electrophoresis.Through sequencing of VP1 gene shows that:DHV-Ⅰvaccine strain VP1 gene and ZI07 strains,AV2111 strains,SY5 strains,XZ strains,SH strains,ZJ strains of nucleotide sequence homology were 99.4%,99.3%,99%,92.3%92.1%, 91.7%,amino acid homology were 100%,99.6%,99.2%,96.6%,96.2%,94.9%。Through analysis of genetic phylogenetic tree,We know DHV-Ⅰvaccine strain VP1 gene and xz strain,SH strain,ZJ strain line in the difference branch,the genetic relationship is distant;and ZI07 strain,AV2111 strains,SY5 strains line in a smaller branch,the genetic relationship is recent.By the nucleotide and amino acid sequence analysis,we can see the nucleotide and amino acid homology of VP1 gene in DHV-Ⅰvaccine strains and domestic DHV-Ⅰstrains are all relatively high,they have various degrees in genetic relationship.3.The true face of the structure of DHV-Ⅰgenome is not entirely known,the composition and function of structural and non-structural protein is needed extensive testing to ultimately.With bioinformatics technology,the physico-chemical property,secondary structure and the potential B cell epitopes of VP1 structural protein were forecasted and analyzed.The results will provide a preliminary theory for the biological research of the VP1 structural protein and the preparation of new vaccines.