家蚕胰凝乳蛋白酶抑制剂基因SCI-SB的原核表达、纯化及活性检测

为进一步研究家蚕胰凝乳蛋白酶抑制剂基因SCI-SB的功能和作用机理,利用RT-PCR技术,从家蚕总RNA中扩增到家蚕SCI-SB基因片段,构建了含有GST标签的融合表达质粒pGEX4T-1-SCI-SB。采用pGEX融合蛋白表达系统,在大肠杆菌BL21中得到以包涵体形式存在的重组GST融合蛋白质,表达量可占菌体蛋白的15%。将包涵体变性、复性处理以后,进一步利用GSTTrapFF亲合层析一步获得了重组蛋白。将该重组蛋白免疫新西兰纯种大白兔,获得效价高的多克隆抗体。胰凝乳蛋白酶活性抑制实验结果表明该重组蛋白具有一定的酶活抑制作用。

Purification and Prokaryotic Expression of the Chymotrypsin Inhibitors From Silkworm

RT-PCR was used to abtain the SCI-SB gene from silkworm. The gene was cloned to the expressing vector pGEX4T-1,then a chymotrypsin inhibitor recombinant expression bacteria was constructed.With the pGEX fusion expression system,we expressed the GST-fusion protein in E.coli BL21,which accounted for approximately 15% of germ proteins. By GSTTrapFF affinity chromatography,GST-fusion protein was obtained. This fusion protein showed certain chymotrypsin inhibitor activity. Immunization of rabbits with fusion protein generated high titer antibodies,measured by ELISA.All this made a good ground for the further study of SCI-SB.