Screening And Identification Of Host Cellular Proteins Interacting With Autographa Californica Multiple Nucleopolyhedrovirus BV/ODV-E26

Autographa californica multiple nucleopolyhedrovirus(AcMNPV), is the representative species of Baculovirus in the baculovirus study. AcMNPV BV/ODV-E26 (E26) is a structural protein of the virus and involved in vesicle membrane composition of BV and ODV. E26 is related to not only the envelope protein, but also with the DNA replication and late gene expression, but its specific mechanism of action is unclear. In this study, we attempt to discover the function of E26 by investigating interactions between E26 and host proteins. The specific results are as follows.(1) Screening the Sf9 host proteins gene that are interacted with AcMNPV E26 from the cell cDNA library. We inserted the e26 gene from AcMNPV genome into the plasmid pGBKT7 to build the bait plasmid pGBKT7-e26. We then used the bait plasmid pGBKT7-e26 to screen the Sf9 host proteins gene from the cell cDNA library by yeast two-hybrid assay, and identified 17 positive clones that are interacted with E26. After sequencing analysis, these positive clones encode four kinds of proteins. They are Arp2/3 complex P21 subunit (P21), serine protease precursor, ribosomal protein S7, and ribosomal protein L40.(2) GST-pull down analysis of the inteacraction between AcMNPV E26 and P21. We constructed the recombinant viruse, which can express the GST-P21 fusion protein and E26-His fusion protein using the Bac-to-Bac system. Then, we used the recombinant viruse to transfect and infect Sf9 cells. GST-pull down analysis showed that E26-His was co-purified with GST-P21 by GST-bind resin. It confirmed that E26 may interact with P21.(3) Bimolecular fluorescence complementation analysis of AcMNPV E26 and P21. We constructed the recombinant viruse, which can express the E26-VC155 (the C-coding sequence of YFP) fusion protein and P21-VN173 (the N-coding sequence of YFP) fusion protein using the Bac-to-Bac system. Then, we used the recombinant viruse to ransfect and infect Sf9 cells. We foud yellow fluorescence was observed by confocal microscopy in the infected cells, and most of the fluorescence accumulated in the nucleus. This further confirmed the interaction between AcMNPV E26 and Sf9 P21 protein.(4) Truncated e26 gene to identify interaction sites between E26 and P21 protein. we constructed 9 truncated e26 mutants then cloned the truncated e26 mutants into plasmid pGBKT7. The interactions between E26 truncated mutants and P21 were tested in the yeast. The experimental results showed that the interaction sites between E26 and P21 protein located between the 76-100aa.(5) The prokaryotic expression AcMNPV E26 protein and the preparation of polyclonal antibodies. To further research for the E26 protein function better,we expressed the E26 in the Prokaryotic bacteria and successfully prepared polyclonal antibody E26. Further analysis showed that the antibody titer antiserum has high specificity and potency.