Molecular Cloning And Experession Of Cyclophilin A And Cyclophilin10in Spodoptera Litura

Cyclophilins(Cyps) which possess peptidyl-prolyl cis-trans isomerase activity, are widely distributed in organisms as constitutive proteins.Cyclophilins also play a key role in a series of biological procedures, such as catalyzing protein folding, attendance in apoptosis, signal transductions and assembling in HIV-1capsid protein. In recent years, the research about Cyps mainly focuses on its role in mammals but it is the first time to research in Spodoptera litra. These results provide valuable information for further exploring the roles of cyclophilin A and cyclophilin10in the insect immune response.SpliCypA and SpliCyp10were cloned from cDNA of Spodoptera litura. The ORF of SpliCypA was498bp and the deduced protein comprised165amino acids with a calculated molecular weight of17.62kDa. SpliCyp10gene included an open reading frame (ORF) of490bp and encoded a protein of161amino acids and its weight was17.89kDa. The SpliCypA gene encoding amino acid sequence homo logy of Spodoptera litura to Rattus norvegicus, Homo sapiens, Xenopus laevis, Bom by xmori and Mythimna separate was74.4%,73.9%,76.8%,83.6%and95.8%, respectively.The prokaryotic expression plasmid pET32a-SpliCypA was constructed successfully and got SpliCypA antibody from the company. In the eukaryotic system, fluorescence microscopy and Western b lot were applied to identify the expression of SpliCypA fusion protein, and the stable expression fusion proteins were identified by Western blot in the four insect cell lines. Image of location of the protein in High Five was obtained by fluorescence microscope. The weight of SpliCypA fusion protein was21.29kDa. The protein was located in cytoplasm. After pIZT/V5-His-SpliCypA transfected into High Five cells, the siRNA mediated CypA silence. The High Five cells which were transfected with pIZT/V5-His-CypA-siRNA-22and pIZT/V5-His-CypA-siRNA-37showed lower expression of CypA. The protein was identified by the specific antibody. The eukaryotic expression plasmid of SpliCyp10was constructed and its recombinant protein weight was21.75kDa. Furthermore the protein was successfully detected in two cells by Western blot after pIZT/V5-His-SpliCyp10plasmid had transfected into Sf9and Spli221cells in96h. The protein was located in cytoplasm especially surrounding the nuclear.