蛇毒锯鳞蝰素基因Leul4－Lys15－Glu16的定点突变

Site-directed Mutagenesis of the Gene of Echistatin Leu14-Lys15-Glu16

本研究的目的是利用蛋白质工程定点突变的方法，在蛇毒锯鳞蝰素基因分子上增加另一个保守序列ＲＧＤ（１４位精氨酸残基，１５位甘氨酸残基，１６位天冬氨酸残基），以其增加该分子的生物活性，并探讨蛋白质一级结构，空间结构和功能的关系。在质粒ｐＪＣ２６４的基础上，利用ＰＣＲ定点突变方法，对蛇毒锯鳞蝰素基因Ｌｅｕ１４－Ｌｙｓ１５－Ｇｌｕ１６进行定点突变，使相应的ＤＮＡ片段变成表达Ａｒｇ１４－Ｇｌｙ１５－Ａｓｐ１６的核苷酸顺序，经酶切和ＤＮＡ测序鉴定正确。ＣＮＢｒ裂解后，用反相ＨＰＬＣ分析，分离制备突变体蛇毒锯鳞蝰素，制备的突变体蛇毒锯鳞蝰素的Ｎ－末端１０个氨基酸残基与天然蛇毒锯鳞蝰素的相同。在人的富含血小板血浆测活体系中，经１０μｍｏｌ／Ｌ的ＡＤＰ诱导，突变体蛇毒锯鳞蝰素的ＩＣ５０为３．０×１０^（－７）ｍｏｌ／Ｌ；重组野生型蛇毒锯鳞蝰素的ＩＣ５０为４．０×１０^（－７）ｍｏｌ／Ｌ；天然蛇毒锯鳞蝰素的ＩＣ_（５０）为２．８×１０^（－７）ｍｏｌ／Ｌ。此外，就蛇毒锯鳞蝰素中保守序列Ａｒｇ－Ｇｌｙ－Ａｓｐ以及其空间构象对蛇毒锯鳞蝰素抑制血小板凝集之间的关系进行了讨论。

In order to probe relationship of inhibition activity of platelet agggregation and RGD conformation beneficial to binding in Echistatin,we used the site-directed mutation technique to install another RGD sequence into one of irregular loops retaining a degree of conformational flexibility and substituting Leu-14,Lys-15,Glu-16 of(Leu-28)Echistatin.The mutant(Arg-14,Gly-15,Asp-16,Leu-28)Echistatin did not lose its inhibition activity of platelet aggregation;however,it showed at least as high activity as(Leu-28)Echistatin,or even a little higher than(Leu-28)Echistatin.Thksuggested that both RGD sequences inserted in one loop with a degree of conformational flexibility.The original RGD(Arg-24,Gly-25,Asp-26)motif projecting significantlly from the surface of the scaffold or core might contribute synergistically to the function of inhibiting platelet aggregation indueed by 10μmol/L ADP(final concentration).These resalts are useful in the elucidation of the relationship of structure and function of Echistatin-like disintegrins and GPⅡ b /Ⅲ-like integrins.