Effect On Larvae Physiological Parameters Of Viruses Infection And Construction Of CBD Mutants For Ha83 Of Helicoverpa Armigera Nucleopolyhedrovirus (HearNPV)

Helicoverpa armigera nucleopolyhedrovirus(HearNPV), is the representative specie of nucleopolyhedrovirus Group II in Baculoviridae, which genome sequence was reported since 2001. Compare to Autographa californica multiple nucleopolyhedrovirus(AcMNPV), which is the model species in NPVs, there were less study on the HearNPV genes. Less than 50% of the HearNPV orfs were well investigated and reported while more than 80% of the AucaMNPV orfs had been well characterized. The ha83 is a chitin binding domain(CBD) encoding gene that has no homologous genes in AucaMNPV.Several recombinant HearNPVs with the nurotoxin genes on the physiological parameters of host larvae were compared with wild type virus. And site-directed mutants of six specified cysteine in CBD of ha83 were constructed. The induced condition of the prokaryotic and eukaryotic expression for both Ha83 and the mutants were optimized and the expression products were purified and identified. The chitinase activity difference between ha83 knock- out HearNPV and the wild-type HearNPV were compared. The results are showed below:1. The average water content of Meeu recombinant HearNPV is significant lower than the other infection group. The glycogen, protein and lipid proportion of RjAa17 f recombinant HearNPV is significant lower than all the other groups, and the average weight of the host larvae is also lower than the other groups. The RjAa17 f recombinant HearNPV causes the apocleisis and paralysis of the infected larvae to reduce the metabolism.2. Successfully constructed the prokaryotic and eukaryotic expression vector for the site-directed mutants of six specified cysteine of the CBD of ha83.3. Successfully expressed the Ha83 and the mutants through prokaryotic and eukaryotic expression, and verified that the mutation did not affect the antigenicity of Ha83.4. A chitinase activity assay was carried out for comparison of ha83 knock-out HearNPV and the wild-type HearNPV. The result showed that after 2 d.p.i., the chitinase activity of ha83 knock- out HearNPV was higher than the wild-type, indicating a possible interaction between Ha83 and the chitinase of HearNPV.