Study On Unknow ORF Of Genome Of White Spot Syndrome Virus In Macrobrachium Rosenbergii

White spot syndrome virus(WSSV), which was the pathogeny of white spot syndromeof prawn, was one of the most dangerous infectivity diseases. The research on pathogeny,epidemiology, diagnostics and mechanism of infection in WSSV has reached to molecularlevel. Three editions of complete sequence of WSSV have released on GenBank. Thekeystone of research in WSSV genomics has been changed to function genomics, namely toanalysis of multitudinous products of gene. At present, the function of 10 genes have beenpreliminary determined, 36 genes was homology to known protein. But still many geneswere unknown.In this research, a special pair of primers was designed based on complete sequence ofwhite spot syndrome virus (NO.AF332093) released from GenBank to amplify a fragmentwith total DNA template from Macrobrachium rosenbergii de man which was suspicious ofbeing infected with WSSV. The fragment then was cloned and sequenced. Blast analysisindicates the fragment belonged to VP28 of WSSV. The result of the dendrogram bycomparing WSSV VP28 from M. rosenbergii with twenty published ones from GenBankdisclosed that the virus from Penaeus. Chinensis,Penaeus. monodon,Penaeus. setiferus,Penaeus. japonicus, and Penaeus. homarus were present, in one phylogenetic branch, andthe one from P. indicus belonged was at another single branch, and the one from M.rosenbergii had farthest relationship comparing with all of them.Simultaneously, five pairs of primers were designed based on unknown gene ofcomplete sequence of white spot syndrome virus (NO.AF332093) released from GenBankto amplify five fragments with total DNA of WSSV. These five fragments were insertedinto plasmid pET-28a (+) respectively. Then WSSV ORF023 and WSSV ORF254 wereanalyzed and their second structure of protein were predicted in via bioinformatics.the pET-28a(+)-WSSV ORF023 and pET-28a(+)-WSSV ORF254 were expressed inE.coli BL21. The result showed that product of WSSV ORF023 was smaller than theprediction. There also had several small bands of WSSV ORF254 expression. It might dueto the rare codons of E.coli existing in WSSV ORF023 and WSSV ORF254. and anamplified fragment named WSSV ORF147 was inserted into pBacPAK-His(1) vector toform an eukaryotic expression vector for further study.