Construction Of The Bac-to-Bac System Of Bombyx Mori Nucleopolyhedrovirus And Functional Analysis Of Bm21

Baculovirus expression vector system has been widely used for production ofrecombinant proteins. In this thesis, the Bac-to-Bac expression system of Bombyxmori nucleopolyhedrovirus (BmNPV) was constructed. The Bac-to-Bac system wasthen improved by deleting the chitinase and v-cathepsin genes of the bacmid toenhance the expression level of heterologous gene. The function of the orf21 ofBmNPV (Bm21) was also analyzed using the Bac-toBac system. The thesis containsfour chapters:Chapter One gave an overview of baculoviruses and the baculovirus expressionvector system(BEVS). The molecular biology of BmNPV and advances of BmNPVBEVS were summarized.Chapter Two described the details of the construction of the BmNPVBac-to-Bac expression system. To construct the bacmid of BmNPV, a transfer vectorwas constructed which contained an Escherichia coli (E. coli) mini-F repticon,kanamycin resistance and a lacZ: attTN7: lacZ cassette within the upstream anddownstream regions of BmNPV polyhedrin gene. Through homologousrecombination in vivo, 17 different genotypes of BmNPV bacmids were screenedand named BmBacJS 1 to BmBacJS 17. One of bacmid colonies, BmBacJS13, whichhad similar restriction enzyme digestion profiles to that of wild-type BmNPV, wasselected for further research. The polyhedrin gene (ph) was inserted into BmBacJS13and the infectivity of BmBacJS13-ph was investigated in vivo or in vitro. The resultsindicated BmBacJS13-ph had the similar infectivities to that of wild-type BmNPV.Furthermore, the egfp gene was introduced into BmBacJS 13 to test the foreign gene expression both in vitro and in vivo. The green fluorescence could be detected inboth infected BmN cells and the tissues of infected B. mori larva. The above dataindicated that a Bac-to-Bac expression system of BmNPV was constructedsuccessfully.In Chapter Three the BmNPV Bac-to-Bac expression system was modified to enhance theexpression of foreign protein. The chitinase and v-cathepsin genes of BmBacJS13 were deleted togenerate BmBacJS 13ACC. The luciferase under signal peptide of AcMNPV chitinase was used asa marker gene and inserted into BmBacJS13 and BmBacJS13ACC, The intracellular and thesecreted Lusiferase activities of both viruses were compared. The results showed the expressionlevels of total and secreted recombinant protein of BmBacJS13ACC-Iuc were significantly higherthan that of BmBacJS13-luc. Therefore, the modified bacmid provides a better tool forrecombinant protein expression.In Chapter Four the function of orf21(Bm21) BmNPV was characterized.Bm21 was predicted to encode a protein of 55.8 kDa. RT-PCR analysis indicated thatthe transcriptional product of Bm21 was first detected at 12 h post infection. Westernblot analysis showed the protein could be first detected from the 36 h post infection.Transient-expression of BM21-EGFP with and without viral infection indicated thatBM21 was localized in the nuclei of the transfected BmN cells. A Bm21 knockoutbacmid BmBacJS13A21 was made and the recombinant virus was examined in BmNcells or insect larvae. The results showed that there no difference in BV growth curveand LC₅₀ between knockout virus and control virus, but the ST₅₀ of the knockoutvirus was about 8 h longer than the parental virus (significant difference). Theseresults indicated that Bm21 was not essential for viral replication but couldaccelerate viral killing in the host insect.