Daxx-C626-740原核细胞表达载体的构建与诱导表达  

Construction and Induced Expression of Prokaryotic Expression Vector of Daxx-C626-740

作　　者：李耀林[1] 周艺[1,2] 何爱桃[1,2] 唐双阳[1] 王蓉[3] 万艳平[1,3] 

机构地区：[1]南华大学病原生物学研究所,湖南衡阳421001 [2]南华大学公共卫生学院,湖南衡阳421001 [3]南华大学护理学院,湖南衡阳421001

出　　处：《中国医药指南》2013年第35期1-3,共3页Guide of China Medicine

基　　金：湖南省教育厅重点项目(11A102);湖南省教育厅一般项目(12C0337);衡阳市科技局项目(2011KJ2)

摘　　要：目的为了研制Daxx-C抗体并观察其在宫颈癌细胞中的应用效果,构建原核表达载体pET28a/DM626-740,将其在大肠杆菌中进行诱导表达,纯化表达产物,鉴定其抗原性。方法用PRIMER5.0软件设计Daxx-C基因片段(氨基酸626-740)特异性引物,引入BamHI和SalI酶切位点,PCR扩增目的基因。将PCR产物连入载体pMD18-T,构建pMD18-T/DM626-740;然后,将BamHI和SalI酶切产物连入载体pET28a,经酶切鉴定及DNA序列分析后,构建原核表达载体pET28a/DM626-740。将其转化E.coli BL21,IPTG诱导表达,利用Ni琼脂糖凝胶层析柱纯化表达产物,Western-blot检测表达物与纯产物。结果双酶切和DNA测序结果显示,PCR正确扩增了Daxx-C基因片段并成功连入载体pET28a,构建了原核表达载体pET28a/DM626-740;该质粒在E.coli中诱导表达分子量约19kDa目的蛋白,且该蛋白能被抗Daxx抗体检出。结论构建的原核表达载体pET28a/DM626-740能在E.coli中表达目的的蛋白6His-DM626-740,并具有良好的抗原性。Objective To prepare Daxx-C antibody and study its used effect on the cervical carcinoma, prokaryotic expression vector of Daxx-C626-740 was constructed and transformed into E.coli, and antigenicity of Daxx-C626-740 purified production was identified. Methods The specific primers of Daxx gene fragment（amino acids 626-740）including BamHI and SalI enzyme sites were esigned by software PRIMER5.0. PCR amplification products were inserted into MD18-T, and the vector of pMD18-T/DM626-740 was constructed. Then, DM626-740 gene fragments from pMD18-T/DM626-740 digested with BamHl and Sall were inserted into pET28a. The prokaryotic expression vector of pET28a/DM626-740 was constructed by identification of enzyme digestion and DNA sequence. The plasmid ofpET28a/DM626-740 was transformed into E. coli BL21. The expression productions were induced with IPTG and purified by Ni-NTA kit. Western bolt was used to analyze expression and purification productions. Results The enzyme digestion and DNA sequencing results showed that Daxx gene fragment（626-740 amino acid residues）was correctly inserted into pET28a. The vector of prokaryotic expression vector of pET28a/DM626-740 was constructed. Conclusion The prokaryotic expression vector of pET28a/DM626-740 constructed can express the aim protein of 6His-DM626-740.The fusion protein of 6His-DM626-740 showed Daxx antigenieity.

关 键 词：DAXX 表达 纯化 抗原性 

分 类 号：R373[医药卫生—病原生物学]