Function Analysis Of SeNPV IE1 In Mammalian Cells

This thesis comprises of four chapters.Chapter 1: The preface is mainly about baculoviruses, baculovirat iel and other viraltransactivating proteins. The introduction to baculovirus includes biologicalcharacters, structures and morphology, replication, application, etc. Then the studyprogress of baculovirus iel including localization and functions was summarized.Finally, several viral transactivation proteins were mentioned.Chapter 2: In this report, we amplified iel gene using SeNPV strain US genome asthe template by PCR, and expressed iel gene in E. coli. Detection and identification ofthe expression by Western blot analysis was done. Another smaller protein wasinduced simultaneously. After in scale expression and purification, we further provedthe expression of ie1, but did not acquired a lot of product. The possible reason is thatthe protein is difficult to express and it is digested when expressed.Chapter 3: In this report, we amplified SeNPV ie1 gene and expressed it by fusing tothe C terminal of enhanced GFP protein in HEK293 cells. Confocal microscopyrevealed that the fusion localized in the nuclear of 293 ceils. After that, the promotersequence of AcMNPV gp64, SeNPV F protein and Drosophila hsp70 was analyzed totest the function of SeNPV IE1 respectively. We found that in the absence of hrsequence, IE1 improved the expression of F promoter but didn't influence gp64promoter significantly, and IE1 also stimulated the hsp70 promoter moderately. Thefurther impact of IE on these promoters in the presence of hr sequence and thelocalization of IE1 in Spodoptera exigua derived cell lines need to be uncovered.Chapter 4: PCR amplification and sequencing results of several genes of strain Z ofSeNPV showed that there's insignificant difference of sequence between at least these genes. But the results suggest that there may be some variance between the twostrains genetically. Restriction endonuclease maps neither differentiated the twostrains. Now someone in our lab is conducting PCR using oligonucleotidesneighboring hr1 to articulate the possible difference.