舌鳞癌侵袭转移相关蛋白CD63-LEL结构域表达及纯化

Expression and purification of CD63-LEL structural domain protein related with invasion and metastasis of tongue cancer

目的构建p ET-29a-CD63-LEL重组质粒,诱导蛋白表达,鉴定表达产物并纯化蛋白,为后续研究CD63-LEL蛋白对人舌鳞癌侵袭转移的影响奠定基础。方法根据美国国立生物技术信息中心(NCBI)中查询到的CD63-LEL基因序列,运用RT-PCR技术从TCA-8113细胞中获得CD63-LEL的c DNA序列,经PCR、双酶切及测序确定获得p ET-29a-CD63-LEL重组载体后,转化至大肠埃希菌E.coli BL21(DE3)感受态中,通过异丙基硫代-β-D-半乳糖苷(isopropylthio-β-Dgalactoside,IPTG)诱导表达,运用镍柱亲和层析法纯化获得p ET-29a-CD63-LEL融合蛋白。结果成功构建了p ET-29aCD63-LEL重组质粒,经SDS-PAGE凝胶电泳检测:在37℃,1 mmol/L IPTG诱导12 h p ET-29a-CD63-LEL蛋白表达量高;在咪唑浓度为MCAC-80/100/200/500条件下可洗脱目的蛋白,Western blot检测Ni-NTA agarose纯化蛋白大小位于26 k U和35 k U处。结论在大肠埃希菌中获得了p ET-29a-CD63-LEL蛋白的大量表达,经镍柱亲和层析可得到较纯的p ET-29aCD63-LEL蛋白。

Objective To construct recombinant vector p ET-29a-CD63-LEL, induce protein expression, identify the expression product and purify the protein, and provide guidance for studying how CD63-LEL affect the invasion and metastasis of tongue squamous carcinoma. Methods The c DNA sequences of CD63-LEL in TCA-8113 cell were detected from NCBI and obtained by RT-PCR, identified by PCR, restriction enzyme digestion and DNA sequencing, and the recombinant plasmids were transformed into E.coli BL21（DE3）. Then, the p ET-29a-CD63-LEL fusion protein was induced by Isopropyl β-D-1-Thiogalactopyranoside（IPTG） and the p ET-29a-CD63-LEL fusion protein was obtained with the method of NI-NTA agarose. Results The recombinant vector p ET-29a-CD63-LEL was constructed successfully. SDS-PAGE detection showed that adding 1mmol/L of IPTG in 37℃ with 12 h induction was the best condition to express p ET-29a-CD63-LEL protein, the protein could be eluted under the condition of imidazole concentration（MCAC- 80/100/200/500）.Western blot detected that the protein was located at 26 k U and 35 k U which was purified by Ni- NTA agarose. Conclusion p ET-29a-CD63-LEL can be largely expressed in E.coli and relatively pure p ET-29a-CD63-LEL protein can be achieved by Ni-NTA agarose purification.