苦瓜MAP30基因的克隆表达及对BGC-823细胞形态的影响  

Cloning and expression of MAP30 gene in Momordica charantia and influence of the morphology on BGC-823 cell

作　　者：黄河[1] 邵世和[2] 韩晓红[2] 田树伟[1] 韩军[1] 黄世腾[2] 

机构地区：[1]江苏大学生命科学院 [2]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2010年第2期136-140,共5页Journal of Jiangsu University：Medicine Edition

基　　金：江苏大学科技创新团队项目(2008-018-02);江苏大学高级人才启动基金项目

摘　　要：目的:克隆苦瓜蛋白MAP30基因,并在大肠埃希菌BL21(DE3)中表达,初步研究MAP30蛋白对BGC-823细胞的影响。方法:应用PCR技术从苦瓜基因组中扩增出MAP30基因片段,将其TA克隆和双酶切鉴定,再进行测序分析,并构建重组表达载体pET-28a-MAP30,转化到表达菌BL21(DE3)中,经IPTG诱导,用NTA-Ni2+树脂分离纯化所表达的MAP30融合蛋白,并经SDS-PAGE电泳鉴定;纯化的蛋白作用于BGC-823细胞后通过光镜和电镜进行形态学观察。结果:成功扩增出MAP30基因为861bp,与基因库中公布的DQ643967同源性为100%,与DQ643968和S79450的同源性为99%;成功构建pET-28a-MAP30原核表达重组质粒;通过NTA-Ni2+树脂分离纯化目的蛋白,获得原核表达的大小约为30000的MAP30融合蛋白,与预测一致;蛋白作用细胞后,细胞形态有明显变化。结论:成功构建pET-28a-MAP30表达载体并获得原核表达的MAP30融合蛋白;重组MAP30蛋白能引起BGC-823细胞明显的形态学变化。Objective：To clone the MAP30 gene and to express it in E.coli BL21（DE3） and to detect the influence of the MAP30 protein on proliferation in BGC-823 cells.Methods：Polymerase chain reaction（PCR） was used to amplify the MAP30 gene from bitter guard genome DNA.Then the target gene was inserted into the vector pGEM-T.The pGEM-T-MAP30 vector was transformed into E.coli DH5α and identified by restriction digestion and DNA sequencing.Then the MAP30 gene fragment was inserted directionally into expression vector pET-28a.The pET-28a-MAP30 expression vector was transformed into E.coli BL21.The his-MAP30 fusion protein was expressed in E.coli BL21 induced by Isopropyl β-D-1-thiogalactoside（IPTG）.The target fusion protein was purified by Ni-column and identified by SDS-PAGE.The morphological change of cell was observed by light microscope and the electron microscope.Results：DNA sequence analysis showed the sequence of MAP30 was 861 bp.It had homology with DQ643967、DQ643968 and S79450.The prokaryotic expression vector pET-28a-MAP30 was efficiently transformed into E.coli BL21.The fusion protein was expressed at 30 ℃ after 0.5 mmol/L IPTG induction for 5 hours.The protein was conveniently purified using NTA-Ni-Charged Resin affinity column with above 90% purity and its relative molecular weight was 30 000.After BGC-823 cells were treated by MAP30,the cells were observed obvious morphological changes.Conclusions：The prokaryotic expression vector pET-28a-MAP30 was constructed successfully and the fusion protein MAP30 was obtained from E.coli BL21.The recombinant MAP30 protein can cause the morphological change of BGC-823 cells significantly.

关 键 词：苦瓜 MAP30 克隆 BGC-823细胞 凋亡 电镜 

分 类 号：R979.1[医药卫生—药品]