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

发布时间：2018-06-21 23:26

  本文选题：凋亡素 + 鸡贫血病毒　； 参考：《中国生物工程杂志》2017年02期

【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。
[Abstract]:In order to obtain the highly soluble chicken anemia virus apoptin gene (CAV-Apoptin), the CAV-Apoptin gene sequence was optimized according to the preference of Escherichia coli codon, and ligated to pBCX vector containing msyB chaperone gene, and transformed into E. coli BL21 (DE3) for induction expression. The protein was purified by nickel column affinity chromatography after SDS-PAGE. The activity of CCK-8 and ladders was observed by microscope. The results showed that the expression vector pBCX-CAV-Apopin was successfully constructed, and a large number of soluble expression products were obtained in E. coli at 37 鈩，

本文编号：2050492