Functional Analysis Of The Baculovirus Chitinase Gene

Baculovims chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. Insects infected with ChiA-deficient remained intact after death. The liquefaction of cadavers is probably critical to the release of progeny virus and its dissemination into the environment. In this paper, the ChiA gene of Helicoverpa armigera single-nucleopolyhedrovirus (HaSNPV) without the N-terminal signal peptide encoding sequence (ChiAS-) was expressed in bacteria and insect cells, respectively. To analyse the function of Bombyx mori nucleopolyhedrovirus (BmNPV) ChiA gene, the gene was deleted. The results may provide some evidences for the embedded research of baculovirus molecular biology and the modification of BmNPV expression system as well as the development of chitinase as a new Bt synergist.1. Expression of HaSNPV ChiA gene in E.coli and insect cells and the synergistism of expressed products with Bt in killing insectsTo investigate the synergistism of baculovirus chitinase with Bt against insect pests and to develop a new Bt synergist, the ChiA gene of HaSNPV was expressed in bacteria and insect cells, respectively. The PCR product of the needing region of mature chitinase e was cloned into the prokaryotic expression vector pET28a and the baculovirus expression vector Bac to Bac, and then was expressed in E.coli BL21 and Trichoplusia ni cell line Tn-5Bl-4, respectively. The expressed target proteins accumulated up to about 15% and 10% of the total cellular proteins, respectively. The total cellular proteins with expressed chitinase were mixed with Bt and fed to the second instar larvae of the silkworm (Bombyx mori). The results showed that the lethal time of the silkworm larvae fed with Bt supplemented with the'chitinase produced in Tn-5Bl-4 cells and in E.coli were much shorter than that of the corresponding controls, LT5o shortened from 93.5 h and 95.1 h to 56.2 h and 67.2 h, respectively. The growth rates of the tested individuals also obviously decreased when compared with their corresponding controls. These results suggested that the recombinant HaSNPV chitinase is a promising Bt synergist.Further SDS-PAGE analysis of the expression product of HaSNPV ChiAS' in E.coli suggested that the expressed ChiAS" existed in inclusion body. Therefore, inclusion body was extracted from E.coli andwashed by detergent. Over 30% expression level was achieved as analyzed by SDS-PAGE. After being purified, the purity of expressed product reached above 90%. The purified HaSNPV ChiAS" was used as antigen to immunize rabbit to produce the polyclonal antibodies against chitinase.The PCR product of HaSNPV ChiAS~ was cloned into the donor vector pFastBacHTe with egfp gene and was expressed in the Bac to Bac baculovirus system. Green fluorescence can be seen clearly under fluorescent microscope. SDS-PAGE showed that there was a specific protein with a molecular of 90 kDa. The fluorescence in Tn-5Bl-4 cells infected with recombinant bacmid mainly located in cytoplasm. 2. Function analysis of BmNPV chitinaseThe upstream and downstream flanking sequence of BmNPV chitinase gene {ChiA) and egfp gene were cloned into pSK (+) plasmid in turn, and the transfer vector pEGFP/ChiA~ DNA was extracted. A recombinant BmNPV, rBmEGFP/ChiA", in which BmNPV ChiA encoding region was replaced by egfp, was constructed by cotransfection with pEGFP/ChiA" DNA and BmNPV genomic DNA. In rBmEGFP/ChiA infected cells, egfp was expressed driven by BmNPV ChiA promoter and the fluorescence could be observed under fluorescent microscope, egfp gene expression phase were detected at 0, 2, 4, 8, 10, 12, .14, 16, 24, 30, 48, 72, 96 h p.i., respectively. Fluorescence can be observed clearly at 8 h p.i. It could be concluded that the promoter of chiA began to drive egfp gene expression between 6 h and 8 h p.i.. Peaks in the fluorescence intensity were observed at 48 h p.i., and kept this level until the cell lysis. These results suggest that BmNPV chiA gene promoter can drive...