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)

[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.