【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。更多还原

【Abstract】 [Objective]To construct lipid transfer protein of Peganum harmala(Ph LTP) gene prokaryotic expression vector,and investigate the antitumor activity in vitro of its expressed product.[Methods]The Ph LTP gene from Peganum harmala leaves was got with reverse transcription polymerase chain reaction(RT-PCR) amplification and cloned into expression vector p ET-30 a to gain p ET-30a-Ph LTP.Recombinant Peganum harmala lipid transfer protein(r Ph LTP) was expressed in E.coli,and purified by nickel column.The expression of r Ph LTP was detected by SDS / PAGE and Western blot.Effect of r Ph LTP on the proliferation of cancer cells assessed by MTT.[Results]Prokaryotic expression vector of Ph LTP was successfully constructed and the recombinant protein which the molecular weight was about 18 k Da was expressed as soluble fusion protein in E.coli.The purified r Ph LTP can significantly inhibit the cervical cancer(He La),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 / m L(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 r Ph LTP has potential clinical applications.更多还原