论文部分内容阅读
[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 a prokaryotic expression vector of rat ATRX antigenic amino acid sequence and lay the foundation for the further preparation of ATRX polyclonal antibody and exploring the role of ATRX protein in HPV16-induced cervical cancer. [Method] The ATRX-C_ (2193-2492) gene fragment was amplified by PCR from IF-GFP-ATRX plasmid and cloned into empty vector p ET30a (+). The recombinant plasmid was transformed into competent E. coli BL21 cells The prokaryotic expression vector p ET30a / ATRX-C_ (2193-2492) was identified by PCR, double enzyme digestion and sequencing. The constructed plasmid pTT30a / ATRX-C_ (2193-2492) was transformed and induced by IPTG. The 6His-ATRX-C_ (2193-2492) protein was purified by nickel ion affinity chromatography. [Result] A 900 bp ATRX-C_ (2193-2492) gene fragment was successfully obtained and the prokaryotic expression vector p ET30a / ATRX-C_ (2193-2492) was constructed. The vector was capable of inducing expression in E. coli at a molecular weight of about 34 k Da protein product. [Conclusion] The prokaryotic expression vector p ET30a / ATRX-C_ (2193-2492) can successfully induce the expression of ATRX-C_ (2193-2492) with a molecular weight of about 34 kDa. The purified product of this protein can be used in the preparation of the subsequent ATRX polyclonal antibody Provide experimental basis.