转移相关基因ABCE1编码蛋白的表达及鉴定  

Expression and identification of ABCE1 encoding protein as matastasis association gene

作　　者：韩旭[1] 田野[1] 田大力[1] HAN Xu TIAN Ye TIAN Da-li(Department of Thoracic Surgery, The 4th Affiliated Hospital of China Medical University, Shenyang 110032, Liaoning, China)

机构地区：[1]中国医科大学附属第四医院胸外科,辽宁沈阳110032

出　　处：《生物医学工程与临床》2017年第3期315-320,共6页Biomedical Engineering and Clinical Medicine

基　　金：国家自然科学基金资助项目(30973502;30170914);辽宁省教育厅科学研究项目(L2013302)

摘　　要：目的构建谷胱甘肽S-转移酶(GST)标签的ATP结合盒转运子E1(ABCE1)基因的原核表达载体pGEX-4T-1-ABCE1,通过转化技术,诱导GST-ABCE1重组蛋白表达。方法通过基因克隆技术构建融合ABCE1基因的原核表达载体,经酶切和测序等方法进行检测。通过转化技术,在E.coli BL21(DE3)中诱导GST-ABCE1重组融合蛋白表达。经酶切,Western blot和质谱测序等方法对该重组蛋白进行鉴定。结果 ABCE1基因片段成功插入pGEX-4T-1原核表达载体中,双酶切和测序后,经Gen Bank blast比对验证,证实ABCE1基因插入载体部位正确,碱基序列正确。凝血酶酶切检测重组蛋白GST-ABCE1得到72 000大小的特异条带,该条带可以被ABCE1抗体识别,质谱检测得到ABCE1蛋白的特异性序列,该序列评分138分(P<0.05)。结论 pGEX-4T-1-ABCE1原核表达载体质粒构建成功,并成功诱导GSTABCE1重组蛋白表达,为继续研究ABCE1在肺癌中的作用机制奠定基础。Objective To construct prokaryotic expression vector of pGEX-4T-1-ABCE1 labeled ATP-binding cassette transporter EI（ABCE1） gene of glutathione S-transferase（GST）, and induce GST-ABCE1 recombinant protein expression by conversion technology. Methods The prokaryotic expression vector of ABCE1 gene was constructed by gene cloning technique, and detected by digestion and sequencing methods. The expression of GST-ABCE1 recombinant fusion protein was induced by E. coli BL21（DE3） by transformation technique. The recombinant protein identified by digestion, Western blot and mass spectrometry was used. Results ABCE1 sequences was successfully inserted into pGEX-4T-1 prokaryotic expression vector, and confirmed ABCE1 gene inserted into vector site correctly and base sequence was correct after digested and sequenced by GenBank. The recombinant protein GST-ABCE1 was detected by thrombin digestion and obtained specific band of 72 000, which identified by ABCE1 antibody, the specific sequence of ABCE1 protein was detected by mass spectrometry, and sequence score 138（P 〈 0.05）. Conclusion The prokaryotic expression vector of pGEX-4T-1-ABCE1 is successfully constructed and induced GST-ABCE1 recombinant protein expression, which lay the foundation for further mechanism study of ABCE1 in lung cancer.

关 键 词：ATP结合盒转运子E1 pGEX-4T-1原核表达载体 重组蛋白表达 

分 类 号：Q78[生物学—分子生物学]