微小隐孢子虫热休克蛋白Hsp90的原核表达与鉴定

Prokaryotic expression and identification of Heat Shock Protein 90 of Cryptosporidium parvum

目的对微小隐孢子虫热休克蛋白Hsp90蛋白进行原核表达与鉴定。方法根据微小隐孢子虫Hsp90基因的CDS区设计引物,PCR扩增长度为2136 bp的Hsp90基因片段,然后将其连接至pET-32a(-)载体,构建重组质粒PET-32a-Hsp90。将重组质粒转化入BL21感受态,使用0.1 mmol/L IPTG诱导重组Hsp90蛋白的表达并进行SDSPAGE分析;采用His标签蛋白填料纯化柱对表达产物进行纯化,采用Western blot鉴定重组蛋白Hsp90的反应原性。结果PCR扩增微小隐孢子虫Hsp90基因片段大小为2136 bp,测序表明扩增基因片段大小正确并成功与PET-32a(-)连接。将PET-32a-Hsp90转化入BL21感受态,经IPTG诱导表达分子质量约为78.32 ku的重组Hsp90蛋白,Western blot检测该蛋白能被Anti-His标签抗体识别。结论成功构建PET-32a-Hsp90重组质粒,并通过原核表达系统成功表达具有反应原性的重组Hsp90蛋白,为该蛋白的功能研究奠定了基础。

Objective To express and characterize the heat shock protein 90(Hsp90)of Cryptosporidium parvum using prokaryotic expression system.Methods Primers were designed according to the CDS region of Hsp90 gene of Cryptosporidium parvum.Hsp90 gene was amplified by PCR with a fragment length of 2136 bp,and ligated into the pET-32a(-)vector to construct the recombinant plasmid PET-32a-Hsp90.Recombinant plasmid was transformed into competent cells BL21(DE3),and its expression product was induced with 0.1mmol/L IPTG.The Hsp90 recombinant protein was initially verified by SDS-PAGE.The expression product was purified using His packed protein purification column.Western blot was used to identify the reactivity of the recombinant protein Hsp90.Results PCR amplified the Cryptosporidium parvum Hsp90 gene fragment as 2136 bp,and sequencing results indicated that the amplified Hsp90 gene fragment was correct and successfully ligated with PET-32a(-).The recombinant plasmid PET-32a-Hsp90 was transformed into competent cells BL21(DE3).IPTG induced expression of recombinant Hsp90 protein with a molecular mass of approximately 78.32 ku.The purified Hsp90 protein was characterized by Western blotting indicated using Anti-His tag antibody.Conclusion This study successfully constructed the recombinant plasmid pET-32a-Hsp90,and the Hsp90 recombinant protein wasexpressed by the prokaryotic system,and its reactogenicity was confirmed by Western blot,which laid the foundation for the functional study of this protein.