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Fusion Expression of Bombyx Mori Baculovirus Polyhedrin Gene and Foreign Target Gene

Author: GuoAiQin
Tutor: WuXiaoFeng
School: Zhejiang University
Course: Special Economic Animal Feeding
Keywords: Bombyx mori BmNPV polyhedrin fusion expression expression level
CLC: Q786
Type: Master's thesis
Year: 2009
Downloads: 73
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Abstract


Polyhedrin was efficiently expressed at very late stage of the baculovirus infection cycle, and was used to occlude virions by forming supramolecular polyhedron. The polyhedrin protein accumulates to very high levels within the infected cell and accounts for 20-50% of the total cell proteins. In the baculovirus expression vector system, foreign genes usually were expressed under the control of polyhedrin promotor, but their expression level were far away from polyhedrin. In this paper, various truncated fragments of BmNPV polyhedrin gene (polh) were cloned, and a series of novel donor vectors which containing polyhedrin promoter and various fragments of above polh gene were constructed. Several recombinant BmNPV viruses were subsequently developed through fusion of those fragments with EGFP which used as a reporter gene. Using thus constructed viruses, we observed whether it could form polyhedra in the affected cells, and whether this fusion expression strategy could improve the expression level of foreign gene. The results were summarized as follows:1. Different BmNPV polh fragments were well expressed in BmN cells, but no polyhedra was observed in the affected cells. This results was different from the previously report, which confirmed that amino acids between 19 and 110 was the required domain for AcMNPV polyhedra supramolecular assembly. This domain of AcMNPV and BmNPV was analysised, the differentia at 26 and 49 amino acids site may significantly affect the formation of polyhedra.2. These novel donor vectors constructed were used to develope recombinant BmNPV viruses fused with EGFP through Bac-to-Bac expression system. Higher level of expression were observed. Higher rate of fluoresced cell and stranger fluorescence intensity than control were observed. The expression level of these fusions were 3 to 5 fold than that of control. Transcription analysis indicated that the level of EGFP mRNA were obviously 2 to3 fold higher than that of control by using RT-PCR. This results demonstrated that the insertion of polh fragment downstream from polyhedrin promotor may play as a positive regulator to the transcription from polyhedrin promotor.The results obtained in this study proved that the fusion expression strategy provided an approach to remarkably improve the expression of foreign gene in Bac-toBac expression system. It will improve the efficiency of Bombyx mori baculovirus expression vector system and developing the silkworm as better bioreactor.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Gene expression
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