Analysis Of Bro Genes From Helicoverpa Armigera Single Nucleocapsid Nucleopolyhedrovirus (HaSNPV)

This thesis includes four chapters.In chapter one, a brief introduction of baculovirus and recent advances in baculovirus repeated ORFs (bros) research were presented. The background and the aim of the thesis were also described.Chapter two is a report on prokaryotic expression of three bro genes from HaSNPV and generation of specific antibodies. Three bro genes were amplified by PCR from the genome DNA of HaSNPV. The PCR fragments were cloned into pBluescript KS(+) plasmids. Then the interested genes were subcloned into expression vector pProExHTb. After the induction of IPTG, the E. coli DH5α which containing bro-a, bro-b and bro-c expressed proteins with molecular weights of 32 kDa,64 kDa and 58 kDa respectively, which were in agreement with the expectation. The purified expressed proteins were used to immune rabbits separately. Western Blot analysis using the multiclonal antibodies derived from the rabbits indicated that these antibodies could react specifically with the target proteins and were suitable to be used for further functional analysis of bro genes. In chapter three, in order to detect the transcription and translation profile of three bro genes, RT-PCR and Western Blot were performed on the HaSNPV infected HzAM1 cells. RT-PCR results showed the transcription of bro-a, bro-b and bro-c began at 6hr p.i., 12hr p.i. and 4hr p.i., respectively. Western Blot analysis indicated that translation of bro-a, bro-b and bro-c began at 24hr p.i., 16hr p.i. and 4hr p.i. respectively. All of the bro genes were transcribed and translated actively up to 96hr p.i.. Transcription and translation of bro-a and bro-b reached a maximal level at 48hrp.i.. In chapter four,we tried to construct bro genes deletion or insertion recombinant HaSNPVs. In order to construct bro-a and bro-b genes deletion mutants, we decided to delete a region of 4.35 kb which contains hr2～bro-a～bro-b～hr3. A transfer vector named pCMM1 was constructed successfully. Two methods were used to generate the recombinant HaSNPV. Method 1, HzAM1 cells were co-transfected with the pCMM1 and w.t. HaSNPV DNA. Method 2, a linear fragment of pCMM1 was introduced to E.coli harboring HaBacHZ8 for homologous recombination. Exhaustive efforts failed to obtain an expected recombinant. Due to the complexity of hr2～bro-a～bro-b～hr3 region, we could not conclude whether this region, hr2～bro-a～bro-b～hr3, is necessary for the replication of HaSNPV. As for bro-c mutant, a positive insertion mutant HaBacEa3 was identified by screening of the existing random insertion mutants library. HaBacEa3 was confirmed by PCR, sequencing and genome enzyme digestion. However, the transfection of HzAM1 with HaBacEa3 failed and it suggested that bro-c might be essential for the replication of HaSNPV.