Daxx-C626-740原核细胞表达载体的构建与诱导表达

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BamHl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Sail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．coliBL21，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。

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

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.