Cloning And Expressional Analysis Of Three Lipase Genes From Anteraea Pernyi

Antheraea pernyi, one of the lepidopteran insects, is an important economic insect. It mainly distributed in the northeast of China, Shandong and Guangxi province. Antheraea pernyi nuclearpolyhedrosis virus (ApNPV) is the pathogen of tussah’s pyogenic disease. The disease has caused large number production cuts. The researches on insect defense mechanisms against pathogenic microorganisms have significantly increased on both molecular and organismic level especially the immune to bacteria has been deeply researched. However, few studies have been done on insect immunity against viruses. So, it is important to research the immunity against viruses of Antheraea pernyi.The Bmlipase-1is protein isolated from the digestive juice of Bombyx mori. It was found that Bmlipase-1has the function of anti-virus and it is also one of the few identifiable anti-viral proteins. Three lipase cDNA sequences were screened from the Anteraea pernyi cDNA library and were designated as Iipasel462(GeneBank accession No.KJ638233), lipase1502(GeneBank accession No.KJ638234) and lipase1725(GeneBank accession No.KJ638235). The three genes were cloned by using the fatbody cDNA of Anteraea pernyi as a template and the full length cDNA of the three genes were1000bp,1000bp and876bp, respectively. The three lipase of Anteraea pernyi all have conserved domains belonged to lipase superfamily.In order to explore the function of lipase gene in the immunity of Antheraea pernyi, real-time quantitative PCR was employed to determine the relative expression levels of the the genes in different tissues of Antheraea pernyi pupae after infection with different pathogens. The results showed that the expression levels of the three genes were all increased, but it is different in different tissues. When the Antheraea pernyi larva were infected with ApNPV, the expression of the lipase genes were employed by real-time quantitative PCR. The relative expression levels of the lipase genes in the fatbody and midgut were all increased in fatbody and midgut. It indicated that the three lipase genes were related with the immunity of Anteraea pernyi to ApNPV.Prokaryotic and eukaryotic expression vectors of lipase genes were constructed. And the genes were expressed in vitro by using E.coli BL21and sf9cells. SDS-PAGE showed that the target gene all had been successfully expressed in the two expression systems. When the genes were expressed in the prokaryotic system, the product existed as inclusion bodies in E. coli BL21. And the recombinant Antheraea pernyi lipase provided a foundation for further studying the function of the protein and preparation of lipase antibody. The lipase of Antheraea pernyi expressed in the eukaryotic system showed lipase enzyme activity. It provided a good basis for further studies on anti-viral function of this protein.