鸡贫血病毒凋亡素基因的可溶性融合表达及抗肿瘤活性分析  

Soluble Fusion-expression and Antitumor Activity Analysis of Apoptin of Chicken Anemia Virus

作　　者：冀君[1] 朱晨晨[1] 许鑫[1] 刘晓[1] 冷超粮[1] 史鸿飞[1] 姚伦广[1] 阚云超[1] 

机构地区：[1]南阳师范学院南阳市兽医生物工程技术研究中心/南阳师范学院昆虫生物反应器河南省工程实验室,南阳473061

出　　处：《中国生物工程杂志》2017年第2期26-32,共7页China Biotechnology

基　　金：河南省国际合作项目(152102410055);高层次人才专项项目(70638)资助项目

摘　　要：为获得高效可溶表达的鸡贫血病毒凋亡素基因(CAV-Apoptin),根据大肠杆菌密码子偏爱性优化CAV-Apoptin基因序列,将其连接到含msyB伴侣基因的pBCX载体上,转化至大肠杆菌BL21(DE3)进行诱导表达,经SDS-PAGE鉴定后应用镍柱亲和层析纯化蛋白质,采用显微镜观察、CCK-8实验与DNA ladders测定其抗肿瘤细胞的活性。结果显示,成功构建大肠杆菌表达载体pBCX-CAV-Apoptin,在37℃正常培养条件下的大肠杆菌中得到大量可溶性表达产物,融合蛋白质分子量大小约为42 kDa,50 ml菌液即可获得1.5 mg左右的纯化重组蛋白,CCK-8实验结果显示纯化后的重组蛋白作用36 h后可对套氏淋巴瘤JEKO-1、REC-1细胞生长产生超过60%的抑制率;显微镜观察与DNA ladder实验证明重组蛋白可诱导JEKO-1、REC-1细胞的凋亡,对HUVEC没有诱导凋亡的作用。免疫印迹分析表明重组凋亡素对JEKO-1细胞诱导了Caspase-3的激活,而对Caspase-8的表达没有影响。研究表明融合蛋白msyB-CAV-Apoptin可达到高效可溶表达,且表达产物具有特异抗肿瘤活性。To obtain high-efficiency soluble expressed apoptin gene of chicken anemia virus,the apoptin gene sequence was optimized based on E.coli codon preference and inserted into pBCX plasmid vector harbored msyB chaperonin,and then transformed into the competent into E.coli BL21（DE3） to induce expression,followed by identification of SDS-PAGE and purified by nickel column affinity chromatography. The activity of anti-tumor was evaluated using microscopic examination,CCK 8 experimental determination and DNA ladders tests. The results showed that the E.coli expression vector pBCX-CAV-Apoptin was successful constructed. A large amount of soluble expression product was obtained in E.coli under the normal cultuered condition at 37 ℃.The fusion protein weighted 42 kDa,and 1. 5 mg of purified recombinant proteins could be obtained from 50 ml bacteria liquid. CCK 8 experiment results showed that the purified recombinant protein could induce the growth inhibition rate over 60% of lymphoma JEKO-1 and REC-1 cells post treated 36 h. Microscope and DNA ladders tests intimated that the recombinant protein could induced the apoptosis of JEKO-1 and REC-1 cells rather than HUVEC cells. Western blotting tests indicated that the recombinant protein activated the expression of caspase-3,but not the cleavage of caspase-8 in JEKO-1 cells. It showed that the fusion protein msyB-CAV-apoptin could achieve efficient soluble expression,and the expressed products had specific antitumor activity.

关 键 词：凋亡素 鸡贫血病毒 融合伴侣 高效可溶性表达 活性分析 

分 类 号：Q789[生物学—分子生物学]