蛇毒蛋白Echistatin的C端突变基因的构建，表达及其活性研究

通过ＰＣＲ定点突变的技术，将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→，Ｔｈｒ４９→Ｖａｌ４９），模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ），以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性。突变的基因重组到表达质粒ｐＪＣ２６４上，经ＩＰＴＧ诱导，以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达，表达量占菌体总蛋白的１５～２０％。ＳｅｐｈａｄｅｘＧ－７５初步纯化该融合蛋白，然后用ＣＮＢｒ裂解，透析，冻干，反相ＨＰＬＣ纯化Ｃ端突变体Ｅｃｓ蛇毒蛋白，Ｎ端十个氨基酸分析与天然的相符，在ＰＲＰ（ｐｌａｔｅｌｅｔ－ｒｉｃｈｐｌａｓｍａ）测活体系中，１０μｍｏｌ／Ｌ的ＡＤＰ诱导，Ｃ端突变体Ｅｃｓ抑制血小板凝聚的活性约为野生型４倍。得到了Ｅｃｓ的Ｃ端突变后使Ｅｃｓ抑制血小板凝聚的活性提高的结果。

The Construction, Expression and Characterization of an Echistatin C-Terminal Mutant

The C-terminal of Echistatin gene was mutated (Ala48→Arg48,Thr49→Val49) by PCR method,to simulate N-terminal Gly-pro-Arg-Val of α-fibrin.This mutated gene was recombined into an expression vector pJC264 and expressed in E.coli as a fusion proteins induced by IPTG. The expression level was up to 15～200% of total cell proteins. Mutant Ecs was liberated from the fusion protein by CNBr and was purified to homogeneity by reverse phase HPLC,as judged by N-terminal sequence analysis. Activity of inhibiting platelet aggregation of the C-terminal mutant Ecs was about 3 times higher than that of recombinant Ecs,suggesting that tetra-peptide Gly-pro-Arg-Val of the C-terminal mutant Ecs plays an important role in inhibiting fibrinogen binding to GPIIb/IIIa receptor on platelet surface.