The Construction Of Heliothis Armingera Single-capsid Nuclear Polyhedrosis Recombinant Virus Lacking Cathepsin

Baculovirus is a kind of double-stranded circle large DNA virus, which has been developed as an environment sound pesticide and a powerful vector of foreign protein expression as well as a potential vector of gene therapy, Revealing the molecular mechanism of viral replicaion,package,and transportation will lead the better understanding of baculovirus itself and enhance its application. Among the genes of baculovirus there are many unnecessary genes,of which viral cathepsin is the one.V-cath gene,called cysteine protease gene, Rawling found it In 1992.From then, scientists had done some in-depth reseach on the structure and protein of v-cath.Preliminary study on the function of v-cath had been done.Infection of larvae by the baculovirus results in liquefaction of insect host, which facilitates the efficient dissemination of virus to the environment. Infection of larvae by Autographa califomica nucleopolyhedrovirus mutants lacking v-cath gene can not result in liquefaction of insect host.It reveals v-cath gene have direct relationship with liquefaction of host and degradation of the organization. The product of v-cath in AcMNPV specially degrades actin of insect cells,which result the collapse of cytoskeleton and the death of the host.HaSNPV is a single capsid nuclear polyhedrosis virus separating from Helicoverpa armigera. The HaSNPV v-cath ORF is 1098 base pairs long,encoding a putative protein of 366 amino acids.The amino acids sequence alignment of eleven baculoviral V-CATHs showed that the primary structure and the catalytic sited of the proteins were conserved.A pholygenetic tree of these V-CATHs was constructed by using maximum parsimony analysis,the result indicated that the v-cath gene of HaS NPV had a different evolutionary history from that of other NPVs.In our research,we constructed the HaSNPV recombinant virus lacking the v-cath gene ,which laid a foundation for the research of the function of v-cath.We took two DNA sequence of about 600 bp length,one is in the upriver of v-cath gene sequence of HaSNPV as the upriver homologous exchange arm,the other is in the downriver of v-cath gene as the downriver homologous exchange arm,designed the primer,and then did PCR ampliation.Then the two sequences were inserted into theplasmid pBluescript KS-. Trasfer vector had been constructed when the report gene LacZ gene (B-galactosidase gene) was inserted the position between upriver homologous exchange arm and downriver homologous exchange arm. Hz cell was cotransfered with Transfer vector and the wild HaSNPV DNA.We got the recombinant virus according to incubation of plaque titrations.Checkup the recombinant virus with PCR ampliation.When the larve was infected with recombinant virus, the liquefaction couldn't be observed.We can conclude that the liquefaction of larve host have direct relationship with v-cath in HaSNPV.