摘要
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 Shigella flexneri GST-IpaH4.5 fusion protein expression vector and induce its expression in E.coli.Methods IpaH4.5 encoding gene was cloned from the genome of Shigella flexneri 2a 301 by PCR and inserted into expression plasmid pGEX-4T-1.The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing,and expressed in E.coli induced by isopropyl-beta D-thiogalactopyranoside（IPTG）.Results The desired GST-IpaH4.5 fusion protein（about 86 000 Mr） was expressed and confirmed by SDS-PAGE and Western blot.Conclusion The successful construction of GST-IpaH4.5 fusion protein expression vector has laid foundation for the further research of purifying GST-IpaH4.5 protein and evaluating its funtion in the pathogenicity of Shigella flexneri.
出处
《中国热带医学》
CAS
2011年第8期911-912,915,共3页
China Tropical Medicine
