重组表达人载脂蛋白(a)羧基末端kringle结构域抑制新生血管  

Recombinant human apolipoprotein(a) carboxyl terminal kringles inhibites angiogenesis

作　　者：申乐[1] 陈保生[2,3] 薛红[2,3] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院麻醉科,北京100730 [2]中国医学科学院基础医学研究所 [3]北京协和医学院基础医学院医学分子生物学国家重点实验室生物化学与分子生物学系,北京100730

出　　处：《基础医学与临床》2013年第6期655-660,共6页Basic and Clinical Medicine

基　　金：国家重点基础研究发展计划(973计划)(G2000056902);国家自然科学基金(30100068)

摘　　要：目的利用毕赤酵母重组表达人载脂蛋白(a)[Apo(a)]羧基末端kringle结构域,明确其抑制新生血管和肿瘤细胞增殖的能力。方法分别构建重组表达Apo(a)羧基末端kringleⅤ结构域(RHAKA)与kringleⅣ10型-krin-gleⅤ结构域(RHAKB)的pPICZαA质粒;转染毕赤酵母X-33分泌表达RHAKA与RHAKB,RHAKs利用His.Tag亲和层析纯化,以及反相高效液相色谱与氨基酸残基测序鉴定;明确RHAKs的糖基化及二硫键形成情况后,利用细胞增殖实验与鸡胚绒毛尿囊膜(CAM)实验检测RHAKs对新生血管和肿瘤细胞增殖的影响。结果毕赤酵母可以大量表达RHAKs,并对RHAKs进行翻译后修饰;RHAKA与RHAKB可以抑制血管内皮细胞的增殖和CAM的新生血管,但对肿瘤细胞增殖无直接的抑制作用。结论利用毕赤酵母高效重组表达人Apo(a)羧基末端kringle结构域可以显著抑制新生血管。Objective To characterize some purified recombinant Apo （a） kringles expressed by Pichia pastoris and to illustrate their antiangiogenic and antitumorogenic capacities. Methods Two recombinant proteins RHAKA （kringle V ） and RHAKB （kringle 1V type 10 and kringle V ） were expressed by Pichia pastoris. Both RHAKA and RHAKB, recombined into pPICZcαA, were secreted by Pichia pastoris X-33. Recombinant proteins were con centrated and dialyzed before His . Tag affinity chromatography. Six amido terminal amino acids of RHAKB were analyzed through sequencing the purified protein from reverse-phase high performance liquid chromatography. We＇ve also illustrated several important characters of recombinant proteins, such as glyeosylation and disulfide bonds formation. Finally, recombinant proteins＇ influence on in vitro cellular proliferation and in vivo angiogenesis of chick embryo chorioallantoic membrane （CAM） were tested. Results Piehia pastoris as an expression host may not only express recombinant proteins at a high level but modify them well. Both RHAKA and RHAKB could inhibit angiogenesis in vitro or in vivo, but no such inhibitory effect was found in cultured carcinoma cells. Conclusions Recombinant Apo（a） carboxyl terminal kringles expressed by Pichia pastoris may inhibit angiogenesis significantly.

关 键 词：载脂蛋白 kringle结构域 毕赤酵母 新生血管 

分 类 号：Q78[生物学—分子生物学]