蛇毒半胱氨酸蛋白酶抑制剂的酵母表达及其对B16细胞体外侵袭的抑制作用  

Expression of Recombinant Snake Venom Cystatin in Yeast Pichia pastoris and Its Effects on B16F1 Melanoma Invasion in vitro

作　　者：万榕[1] 宋军[1] 郑海音[1] 张晓艳[1] 林旭[1] 林建银[1] 

机构地区：[1]福建医科大学分子医学中心,福州350004

出　　处：《中国生物工程杂志》2007年第7期21-26,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30371747);福建省科技重大项目(2002Y003)

摘　　要：目的:建立毕赤酵母表达质粒pPICZαA-cystatin,转化酵母细胞生产重组蛋白,探讨蛇毒半胱氨酸蛋白酶抑制剂(sv-cystatin)对肿瘤侵袭的生物学作用。方法:利用PCR扩增技术从pUC18/sv-cystatin质粒中扩增sv-cystatin cDNA并克隆至酵母表达载体pPICZαA上,构建重组质粒pPICZαA-cystatin电激转化Pichia pastori酵母细胞GS115,经1%甲醇诱导获得稳定表达的重组蛋白,改良Boyden小室分析重组sv-cystatin蛋白处理对B16F1细胞体外侵袭力的影响。结果:SDS-PAGE检测和Western blot分析显示分泌表达的sv-cystatin重组蛋白相对分子量约为14kDa,摇瓶发酵每升发酵培养上清可获得16mg的重组蛋白,经亲和层析纯化获得的sv-cystatin重组蛋白具有抑制木瓜蛋白酶的活性。改良Boyden小室实验结果显示:经0.5mg/ml浓度的重组蛋白处理的B16F1细胞穿过Matrigel的细胞数明显低于对照组(52.60±4.58,106±5.9,P<0.01),抑制率为50%。结论:成功实现sv-cystatin的酵母表达,初步证明sv-cystatin重组蛋白可抑制小鼠B16F1细胞体外侵袭作用。To investigate the biological role of snake venom cystatin （sv-cystatin） in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZαA. The linearized recombinant plasmid pPICZαA-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut^＋ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 kDa and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly （ P 〈 0. 01 ）. The data provides the first evidence that sv-cystatin could not only inhibit papain activity, but also suppress the matrigel invasion of B16F1 melanoma cell line.

关 键 词：蛇毒半胱氨酸蛋白酶抑制剂 毕赤酵母 分泌表达 B16F1细胞 侵袭 

分 类 号：Q78[生物学—分子生物学]