十二指肠贾第虫α-6贾第素的原核表达及纯化

Prokaryotic expression and purification of α-6 Giardin of Giardia duodenalis

目的原核表达并纯化十二指肠贾第虫α-6贾第素(α-6 Giardin)。方法以十二指肠贾第虫的c DNA为模板,PCR扩增α-6 Giardin基因片段,连接至p MD18-T载体,得到重组克隆质粒p MD18-T-α-6,将双酶切后得到的目的片段连接至p ET-28a表达载体中,得到重组表达质粒p ET-28a-α-6,转化至E.coli Rosetta感受态细胞中,IPTG诱导表达目的蛋白α-6 Giardin,收集菌体,离心后进行SDS-PAGE及Western blot鉴定,并利用Ni-NTA亲和层析柱纯化融合的α-6 Giardin蛋白。结果质粒p ET-28a-α-6经双酶切鉴定,证明构建正确;表达的目的蛋白α-6 Giardin为带His标签的包涵体蛋白,相对分子质量约40 000,可与鼠抗His单抗特异性结合,具有良好的反应原性;纯化的重组α-6Giardin融合蛋白相对分子质量约40 000,纯度可达80%。结论成功克隆、表达并纯化了十二指肠贾第虫α-6Giardin蛋白,为十二指肠贾第虫α-6 Giardin进一步的功能研究奠定了基础。

Objective To express the α-6 Giardin of Giardia duodenalis in prokaryotic cells and purify the expressed product. Methods Using the c DNA of G. duodenalis c DNA as a template,α-6 Giardin gene fragment was amplified by PCR and cloned into vector p MD18-T. The constructed recombinant plasmid p MD18-T-α-6 was digested with Eco RⅠand Hind Ⅲ,and the obtained target fragment was cloned into expression vector p ET-28 a. The obtained recombinant plasmid p ET-28 a-α-6 was transformed to competent E. coli Rosetta cells and induced with IPTG. The expressed α-6 Giardin was identified by SDS-PAGE and Western blot,and purified by Ni-NTA chromatography. Results Restriction analysis showed that recombinant plasmid p ET-28 a-α-6 was constructed correctly. The expressed α-6 Giardin was a His-tagged inclusion body protein with a relative molecular mass of about 40 000,which showed specific binding to anti-His monoclonal antibody,indicating a good reactogenicity. The purified α-6 Giardin fusion protein,with a relative molecular mass of about 40 000,reached a purity of 80%. Conclusion The α-6 Giardin of G. duodenalis was successfully cloned,expressed and purified,which laid a foundation of further study on the function of α-6 Giardin.