Cloning And Prokaryotic Expression Of SCI-SB From Silkworm

Based on SCI-SB sequence data, RT-PCR technique was used to obtain the SCI-SB gene from silkworm(qingsongxhaoyue). The gene was subcloned into the expressing vector pGEX4T-1.The result of PCR and BamHI/XhoI showed that the designed fragment was amplified correctly.The insertion of the resultant recombinant plasmid pGEX4T-1-SCI-SB was sequenced , a chymotrypsin inhibitor recombinant expression bacteria was constructed.The recombinant plasmid was transformed into E.coli BL21.After inducing by IPTG, the expression production experiment showed that fusion protein GST-SCI-SB was expressed highly in 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 (inhibitor ratio>60%) and typsin inhibitor activity(inhibitor ratio>40%). Immunization of rabbits with fusion protein generated high titer (1.56×10^-4) polyclonal antibodies, measuring by ELISA. The GST-SCI-SB was detected by Western blotting with the polyclonal antibodies.All this made a good ground for the further study of SCI-SB.