融合蛋白AnxB1-MLT的表达、纯化及其对肝癌细胞SMMC7721和HepG2增殖的抑制作用  

Expression and purification of AnxB1-MLT chimera and its inhibitory effect on SMMC7721 and HepG2 cell proliferation

作　　者：王玉招[1] 颜宏利[1] 唐冠楠 林恒荣 章意亮[1] 孙树汉[1] 

机构地区：[1]第二军医大学基础部医学遗传学教研室,上海200433

出　　处：《第二军医大学学报》2013年第11期1171-1176,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81272280);上海市浦江人才计划~~

摘　　要：目的以膜联蛋白B1（AnxBl）为导向分子，与蜂毒素（MLT）基因融合，制备AnxBl一MLT融合蛋白，探讨其对磷脂酰丝氨酸脂质体的结合活性及对肝癌细胞SMMC7721和HepG2增殖的抑制作用。方法利用重叠延伸PCR技术构建AnxBl和MLT的融合基因AnxBl-MLT，克隆至原核表达载体pGEX-5T，转化至宿主菌K802，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导AnxBl-MLT融合蛋白表达，优化表达条件，用谷胱甘肽一孓转移酶（GST）亲和色谱纯化柱纯化融合蛋白。采用磷脂结合实验验证AnxBl-MLT融合蛋白的钙依赖性磷脂结合活性，采用CCK-8方法分别检测AnxBl-MLT融合蛋白对肝癌细胞系SMMC7721和HepG2细胞增殖的影响。结果AnxBl-MLT融合蛋白在宿主菌K802中能够表达，在菌液生长至D600为0．6时，加入诱导剂IPTG至终浓度0．2mmol／L，低温（22~24℃）诱导表达4h，可使蛋白表达量较高且在上清有表达。通过GST亲和色谱纯化柱纯化融合蛋白，SDS-PAGE结果显示在63000处可见单一目的条带，纯度〉95％。AnxBl-MLT融合蛋白保留了AnxBl的钙依赖性磷脂结合活性，并能显著抑制肝癌细胞系SMMC7721和HepG2的增殖。结论成功制备了AnxBl-MLT融合蛋白，该融合蛋白具有AnxBl的钙依赖性磷脂结合活性，可抑制肝癌细胞SMMC7721和HepG2的增殖，为进一步利用动物实验研究AnxBl-MLT的抗肿瘤效果奠定了基础。Objective To obtain a chimera composed of annexin B1 （AnxB1） and melittin （MLT） and to investigate its inhibitory effect on phosphatidylserine liposome activity and SMMC7721 and HepG2 cell proliferation. Methods A fusion gene AnxB1-MLT was constructed by overlap extension gene splicing and then was inserted into plasmid pGEX-ST. The recombinant plasmid was transformed into E. coli strain K802 and induced by IPTG at low temperature. The expression condition was optimized and GST affinity chromatography column was used for purification. Calcium-dependent phospholipid binding assay was used to determine whether the chimera kept the activity of AnxB1. CCK8 analysis was employed to investigate the effect of AnxB1-MLT on 8MMC7721 and HepG2 cell proliferation. Results After being inserted into the expression plasmid, AnxB1-MLT protein expressed in K802 cells, with a high level recombinant protein induced by 0.2 mmot/ L IPTG at 22-24~C for 4 h. The protein AnxB1-MLT was purified by using GST affinity chromatography columri （a band at 63 000） and the purification of the final purified protein was 〉95%. AnxB1-MLT was able to bind to phosphatidylserine liposome in a calcium-dependent manner. CCK8 analysis indicated that AnxB1-MLT inhibited SMMC7721 and HepG2 cell proliferation at a dose-dependent manner. Conclusion We have successfully constructed a chimera AnxB1-MLT, which retains the calcium-dependent phospholipid binding activity of AnxB1, and can inhibit the proliferation of hepatic cancer cell lines SMMC7721 and HepG2.

关 键 词：膜联蛋白B1 蜂毒素 细胞增殖 靶向药物治疗 

分 类 号：R735.7[医药卫生—肿瘤]