原核表达载体pET30a／ATRX-C2193-2492的构建与诱导表达  

Construction and inducible expression of prokaryotic expression vector pET30a/ATRX-C_(2193-2492)

作　　者：唐双阳[1] 刘志敏[1] 李冉辉[1] 申海艳[1] 赵兰华[1] 蔡恒玲[1] 万艳平[1] 

机构地区：[1]南华大学,病原生物学研究所特殊病原体防控湖南省重点实验室,湖南衡阳421001

出　　处：《生物技术》2016年第6期516-520,共5页Biotechnology

基　　金：国家自然科学基金项目(“人乳头瘤病毒16型E2蛋白对Daxx调控细胞凋亡的影响”,No.81402169);湖南省教育厅重点项目(“Daxx在ND10抗HPV16感染中的作用及机制研究”,No.15A164);特殊病原体防控湖南省重点实验室资助项目(湘科计字[2014]5号);湖南省分子靶标新药研究协同创新中心资助项目(湘教通[2015]351号)

摘　　要：[目的]构建大鼠ATRX抗原端氨基酸序列的原核表达载体,为进一步制备ATRX多克隆抗体以及探讨ATRX蛋白在HPV16致宫颈癌中的作用奠定基础。[方法]以IF-GFP-ATRX质粒为模板,PCR扩增获得ATRX-C_(2193-2492)基因片段,并克隆至p ET30a(+)空载体上,转化E.coli BL21感受态细胞,构建原核表达载体p ET30a/ATRX-C_(2193-2492),经PCR、双酶切以及测序鉴定。将构建好的质粒p ET30a/ATRX-C_(2193-2492)转化,经IPTG诱导表达,利用镍离子亲和层析法纯化6His-ATRX-C_(2193-2492)蛋白。[结果]成功获得900 bp的ATRX-C_(2193-2492)基因片段并构建了原核表达载体p ET30a/ATRX-C_(2193-2492),且该载体能在E.coli中诱导表达分子量约34 k Da蛋白产物。[结论]原核表达载体p ET30a/ATRX-C_(2193-2492)能成功诱导表达分子量约34 k Da的ATRX-C_(2193-2492)蛋白,该蛋白纯化产物能为后续ATRX多克隆抗体的制备提供实验基础。[ Objective ] To construct the prokaryotic expression vector of the ATRX - C terminal, for the preparation of its poly- clonal antibody,so as to lay the ground for further explore the role of ATRX protein in cervical cancer caused by HPV16. [ Methods] Using the IF- GFP- ATRX as template,ATRX- C2193-2492 gene was amplified by PCR and then cloned into prokaryotic expression vector pET30a（ ＋ ）. The recombinant plasmid was transfected into E. coli BL21 competent cell, and identified by DNA sequencing , restriction enzyme digestion and PCR assay. The identified recombinant plasmid was transformed into E. coli BL21. The recombinant ATRX - C2193 -2492 protein was induced by IPTG and purified by Ni - NTA affinity column. [ Results] Successful The ATRX - C2193-2492 gene product was obtained to construct the recombinant prokaryotic expression vector pET30a/ATRX - C2193 -2492 successfully. And the recombinant vector can be induce by IPTG to express a kind of fusion proteins with the molecular weight about 34 kDa in E. coli BL21. [ Conclusion] The recombinant prokaryotic expression vector pET30a/ ATRX - C2193-2492 can be induce to express ATRX - C2193-2492 proteins with the molecular weight about 34 kDa, and this purified His - ATRX - C2193 -2492 protein can be used to produce ATRX - C polyclonal antibody.

关 键 词：ATRX 原核表达载体 诱导表达 蛋白 

分 类 号：Q812[生物学—生物工程]