骆驼蓬脂转移蛋白基因克隆、表达及体外抗癌活性分析  

Cloning and expression of lipid transfer protein gene from Peganum harmala and the anti-tumor activity in vitro of its expressed product

作　　者：赵晶[1] 李轶杰[1] 罗晶晶[2] 彭永玉[1] 吕慧[1] 刘东亮[1] 孙素荣[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046 [2]新疆医科大学,新疆乌鲁木齐830011

出　　处：《生物技术》2014年第6期26-31,共6页Biotechnology

基　　金：新疆维吾尔自治区科技支疆项目("新疆骆驼蓬蛋白抗肿瘤作用的研究";No.20091129);新疆生物资源基因工程重点实验室开放基金项目("新疆骆驼蓬抗肿瘤蛋白活性的研究";No.XJDX0201-2010-06)资助

摘　　要：[目的]构建骆驼蓬脂转移蛋白(Ph LTP)原核表达载体,并研究表达产物的体外抗癌活性。[方法]采用RT-PCR法从骆驼蓬(Peganum harmala)叶中扩增Ph LTP基因,将其成熟肽片段克隆至p ET-30a载体上,构建表达载体p ET-30a-Ph LTP,将重组质粒转化E.coli BL21进行原核表达,通过镍柱纯化获得重组骆驼蓬脂转移蛋白(r Ph LTP),经SDS-PAGE和Western blot分析鉴定,用MTT法检测其体外抗癌活性。[结果]成功构建骆驼蓬脂转移蛋白c DNA表达载体p ET-30a-Ph LTP,其在E.coli中以可溶性融合蛋白形式表达,分子量约为18 k Da。经镍柱纯化后重组蛋白r Ph LTP能够显著抑制宫颈癌He La、黑色素瘤B16和食管癌Eca-109细胞的生长,IC50值分别为16.76±1.23、38.92±2.16和9.01±1.01μg/m L。[结论]克隆了骆驼蓬脂转移蛋白基因并在原核表达系统中成功表达,r Ph LTP所显示的显著抗癌活性预示其具有临床应用的潜能。[ Objective ] To construct lipid transfer protein of Peganum harmala （PhLTP） gene prokaryotic expression vector, and investigate the antitumor activity in vitro of its expressed product. [ Methods ] The PhLTP gene from Peganum harmala leaves was got with reverse transcription polymerase chain reaction （RT -PCR） amplification and cloned into expression vector pET- 30a to gain pET- 30a- PhLTP. Recombinant Peganum harmala lipid transfer protein （rPhLTP） was expressed in E. coli, and purified by nickel column. The expression of rPhLTP was detected by SDS/PAGE and Western blot. Effect of rPhLTP on the proliferation of cancer cells assessed by MTT. [ Results ] Prokaryotic expression vector of PhLTP was successfully constructed and the recombinant protein which the molecular weight was about 18 kDa was expressed as soluble fusion protein in E. coli. The purified rPhLTP can significantly inhibit the cervical cancer （ HeLa）, esophageal carcinoma （Eca -109） and melanoma （B16] cells growth,and the value of IC50 was 16.76 ± 1.23,38.92 ± 2.16 and 9.01 ± 1.01 μg/mL （48 hours）, respectively. [ Conclusion] The lipid transfer protein gene of Peganum harmala was cloned and successfully expressed in E. coli. The purified recombinant protein showed the antitumor activity, all showed that the rPhLTP has potential clinical applications.

关 键 词：重组骆驼蓬脂转移蛋白 克隆和表达 纯化 抗肿瘤作用 

分 类 号：Q71[生物学—分子生物学] Q255