The Research Of Cysteine Protease Inhibitor (BmCPI) In Silkworm, Bombyx Mori

Cysteine proteases were mainly considered as degrading enzymes in vivo proteolysis, which were involved in the process of apoptosis, tissue degradation, immune defense and other processes in organism. Silkworm (Bombyx mori), one of the insects, has a special research value due to its ecomomical pupose. Cysteine proteases were involved in the process of silkworm metamorphosis. Their inhibitor, cysteine protease inhibitor in the silkworm (BmCPI) as a regulatory factor of cysteine protease also plays an important role in silkworm development and metamorphosis.Cysteine protease inhibitors were widely distributed in animals, plants and microorganisms, which occupied the active site of cysteine proteases to form a tight complex, thereby inhibiting the activity of cysteine proteases. Until now, fewer information of cysteine protease inhibitor was reported in the silkworm. This study is to identify cysteine protease inhibitor of silkworm (Bombyx mori) and to investigate the expression characteristics and the regulation mechanism through phylogenetic analysis, gene cloning, prokaryotic expression, western Blot, immunohistochemistry and Dual luciferase reporter analysis. Site-directed mutagenesis and other methods, were thereby used to provide data for further investigation on the inhibitor functions in the development process of molting and metamorphosis in silkworm. The main findings are as follows:1. The Expression, Purification, and Antibody Preparation Characterization of BmCPIBased on the SilkDB and NCBI, we found two genes code 129 inhibitor, named BmCPI39 and BmCPI40. BmCPI39 encoded 105 residues protein with predicted molecular weight of 11.16 KD, predicted isoelectric point of 6.09. BmCPI40 encoded 121 residues protein, with predicted molecular weight of 13 KD, and isoelectric point of 4.39. Both the mature form did not contain cysteine and disulfide bonds. Evolutionary analysis showed BmCPI39 and BmCPI40 was closed to the 129 domain of cysteine proteases of BmNPVBmCPI39 and BmCPI40 were cloned and prokaryotic expressed the supernatant of cells. After purification, the two purified proteins were performed to produce polyclonal rabbit antibodies.The titers of BmCPI39 and BmCPI40 antibodies were more than 1:512,000, and the concentrations were 1.62 mg/mL and 1.25 mg/mL, respectively. So the antibodies of BmCPI39 and BmCPI40 could be used for following experiments.2. Identification of activity and key amino acid for recombinant BmCPIBmCPI39 and BmCPI40 activity assay showed that BmCPI39 and BmCPI40 could inhibit cathepsin L activity, even after heat-treated in 80℃. Circular dichroism (CD) scans suggested that BmCPI39 and BmCPI40 had high thermal resilience. The predicted 3D structure of BmCPI39 and BmCPI40 by bioinformatic tool suggested the arginine residues in position 45 could play an essential role in maintaining stability and activity of BmCPI39, which was validated by site-directed mutagenesis analysis.3. Expression characteristics and tissue localization of BmCPISpatio-temporal expression analysis by RT-PCR and Q-PCR show BmCPI39 was mainly expressed in the hemolymph of silkworm and BmCPI40 was mainly expressed in the epidermis of silkworm. The level of expression of BmCP139 in the pupal stage was significantly higher than the larvae stage, while the amount of BmCPI39 expression was significantly increased during molting and metamorphosis. BmCPI40 had opposite expression pattern. The level of BmCPI40 expression in the larvae stage was significantly higher than level of the pupal stage, while the amount of BmCPI40 was reduced during molting and metamorphosis. The pattern of BmCPI39 and BmCPI40 in hemolymph and epidermis showed the similar pattern with the nucleic acid level by Western Blot. The Immunofluorescence analysis showed BmCPI40 was synthesized in the epidermis, then secreted into the skin.4. Preliminary investigation the expression regulation of BmCPIThe 20E induced experiments were performed in silkworm to revalidate increased expression level of BmCPI39 in hemolymph and reduced expression level of BmCPI40 in epidermis. The result showed level of BmCPI39 and BmCPI40 could be regulated by ecdysone. Dual luciferase reporter analysis also showed, promoter activity of BmCPI39 and BmCPI40 was inhibited by 20E after cell treated by 20E.