家蚕Kazal型丝氨酸蛋白酶抑制剂基因BmSPI3的克隆与表达特征分析

Kazal型丝氨酸蛋白酶抑制剂(SPI)在机体的发育、免疫等生理过程中发挥重要的作用。从家蚕蛹cDNA文库中获得一条全长为728bp的基因序列,对该基因编码的氨基酸序列进行同源性比对,发现其蛋白具有保守的Kazal型丝氨酸蛋白酶抑制剂结构域(CⅠ-X3-CⅡ-X8-CⅢ-X14-CⅣ-X6-CⅤ-X13-CⅥ),且P1活性位点为赖氨酸(K),因此将该基因命名为BmSPI3(Gen-Bank登录号:DN237641)。通过PCR扩增BmSPI3基因,经BamHⅠ和XhoⅠ双酶切后成功构建了重组表达质粒pGEX-4T-1-BmSPI3,并转化到E.coliBL21(DE3)表达,经SDS-PAGE电泳检测在约36kD处有明显的蛋白表达条带。提取各发育时期蚕体和5龄幼虫各个组织的总RNA进行荧光定量PCR,检测结果表明:BmSPI3在5龄幼虫期表达量最高,卵期最低;在5龄幼虫表皮中的表达量最高,在马氏管的表达量最低。推测BmSPI3对胰蛋白酶和类胰蛋白酶具有抑制作用。

Molecular Cloning and Expression Profile of a Kazal-type Serine Protease Inhibitor Gene (BmSPI3) from Bombyx mori

Kazal-type serine protease inhibitors play important roles in physiological processes such as development and immune response. We have cloned a full length cDNA (728 bp) sequence from cDNA library of Bombyx mori pupae. Amino acid sequence analysis revealed that its encoded amino acids contained a single domain with a conserved sequence of CⅠ-X3-CⅡ-X8-CⅢ-X14-CⅣ-X6-CⅤ-X13-CⅥ similar to that of Kazal-type serine protease inhibitors,and its P1 active site was lysine (K). Therefore,this gene was designated as BmSPI3 (GenBank accession number DN237641). After being amplified and digested with BamHⅠ and XhoⅠ,an expression plasmid named pGEX-4T-1-BmSPI3 was successfully constructed for the target gene. The expression plasmid was transformed into E. coli BL21 (DE3) cells for expression. SDS-PAGE identified an obvious protein band of about 36 kD. Fluorescence quantitative PCR analyses on expression levels in different developmental stages and different tissues of the 5th instar larvae indicated that BmSPI3 expression was the highest in the 5th instar larvae and the lowest in egg stage. Moreover,among various tissues from the 5th instar larvae,the epidermis had the highest expression level and the Malpighian tubule had the lowest. It is suggested that BmSPI3 has inhibitory effect on trypsin and tryptase.