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目的构建痢疾杆菌GST-IpaH4.5融合蛋白表达质粒,并在大肠杆菌(E.coli)中诱导表达。方法以痢疾杆菌福氏2a 301株全基因组为模板,PCR扩增痢疾杆菌ipaH4.5基因,在ipaH4.5的上游加上BamHⅠ酶切位点,下游加上XholⅠ酶切位点,将ipaH4.5基因定向插入质粒pGEX-4T-1中,构建原核表达质粒pGEX-IpaH4.5并转化E.coliDH5α,筛选阳性重组子,限制性内切酶切鉴定和DNA序列测定,DNA序列正确后提取质粒转化E.coli BL21,筛选阳性转化子,异丙基硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot鉴定表达结果。结果成功构建IpaH4.5原核表达质粒pGEX-IpaH4.5并表达出大小约86 000Mr的GST-IpaH4.5融合蛋白。结论 GST-IpaH4.5融合表达载体的构建,为进一步纯化IpaH4.5蛋白和研究其在痢疾杆菌致病机制中的作用奠定了基础。
Objective To construct the expression plasmid of Shigella dysenteriae GST-IpaH4.5 fusion protein and induce its expression in E.coli. Methods The genomic DNA of Shigella flexneri 2a 301 was used as a template to amplify ipaH4.5 gene of Shigella flexneri. The upstream of ipaH4.5 was added with BamH Ⅰ restriction site and the downstream of Xhol Ⅰ enzyme digestion site. 5 gene was inserted into plasmid pGEX-4T-1, and the prokaryotic expression plasmid pGEX-IpaH4.5 was constructed and transformed into E.coli DH5α. The positive recombinant was screened by restriction endonuclease digestion and DNA sequencing. After the DNA sequence was correct, the plasmid The recombinant plasmid was transformed into E.coli BL21. The positive transformants were screened and induced by isopropylthiogalactoside (IPTG). The expression results were confirmed by SDS-PAGE and Western blot. Results The IpaH4.5 prokaryotic expression plasmid pGEX-IpaH4.5 was successfully constructed and a GST-IpaH4.5 fusion protein of about 86 000Mr in size was expressed. Conclusion The construction of GST-IpaH4.5 fusion expression vector lays the foundation for further purification of IpaH4.5 protein and its role in the pathogenesis of dysentery bacillus.