叶酸受体α原核载体的构建及表达  

Construction and Expression of Prokaryotic Expression Vector Encoding Folate Receptor α

作　　者：张梅[1] 邱郑[1] 邢黎军[1] 强旭 张娟[1] 王旻[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2016年第1期26-29,共4页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(No.81301902);江苏高校优势学科建设工程资助项目

摘　　要：PCR法扩增叶酸受体α基因片段,经NcoⅠ和EcoRⅠ双酶切后克隆到原核表达载体pet22b,并进行双酶切和测序鉴定;将测序正确的重组质粒通过Ca Cl2法转入BL21(DE3)表达菌株,IPTG诱导蛋白表达,镍柱亲和纯化,通过SDS-PAGE,Western blot和ELISA鉴定蛋白表达;免疫小鼠,血清效价检测蛋白免疫原性。经双酶切和测序验证,成功构建FRα-pet22b表达载体。Western blot验证表明,叶酸受体α基因能在BL21中表达目的蛋白,但ELISA值较低。经免疫后小鼠血清与市售叶酸受体α亲和力差,预示原核表达虽能得到叶酸受体α,但其构象可能与真核表达蛋白不同。The c DNA fragment encoding folate receptor α（ FRα） was amplified using PCR and was cloned into the prokaryotic expression vector pet22 b between the two restriction enzyme sites Nco Ⅰ and EcoR Ⅰ. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing. BL21（ DE3） was transformed with the recombinant plasmid using Ca Cl2 method. The expression of FRα was purified by Ni·column and was identified by SDS-PAGE,Western blot and ELISA. The purified protein was injected intramuscularly into BALB / c mice. The immunogenicity of the protein was tested by serum titer. The prokaryotic expression vector was successfully constructed. The expression of FRα in BL21 was confirmed by Western blot,but it showed lower ELISA values,indicating that although the prokaryotic expression can get folate receptor α,its conformation differs from the natural product.

关 键 词：叶酸受体Α 构建 原核表达 蛋白鉴定 亲和力 构象 

分 类 号：Q51[生物学—生物化学]