摘要
目的构建大鼠钙结合蛋白S100A9的原核表达质粒并获得纯化重组蛋白。方法根据大鼠S100A9基因mRNA序列,设计PCR引物,常规扩增后重组连入原核表达载体pET32a,并进行序列测定。利用异丙基硫代-β-D-半乳糖苷(IPTG)和不同温度诱导表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定结果,并亲和层析纯化重组蛋白。结果 PCR扩增获得的条带与预期的DNA表达片段大小一致,克隆构建了pET-32a-S100A9原核表达载体,测序结果与预期完全一致,经亲和层析纯化获得毫克级纯化重组蛋白,并发现该蛋白在21℃有比较强的表达。结论为进一步探讨S100A9蛋白生物学功能奠定基础。
Objective To construct the prokaryotic expression plasmid of rat calcium-binding protein S100A9 and obtain purified recombinant protein. Methods Based on the mRNA sequence of rat calcium-binding protein S100A9,PCR primer pair were designed. The gene was amplified and inserted into pET32a prokaryotic expression vector. After confirmatory sequencing,expression of S100A9 protein was induced by isopropy-β-D-thiogalactoside（IPTG） at different temperatures. The protein was then identified by polyacrylamide gel electrophoresis（SDS-PAGE） and purified by affinity chromatography. Result The results of PCR and the sequence of recombinant plasmims pET-32a-S100A9 were consistent to the expected ones. SDS-PAGE and western blotting showed that the relative molecular weight of the expressive product was same to the expected value. The affinity chromatography purification obtain mg grade purification,and found that the recombinant proteins in the 21 ℃ proteins have a strong expression. Conclusion This study has laid a foundation for further research of biological function of S100A9.
出处
《中国医药生物技术》
CSCD
2010年第6期405-409,共5页
Chinese Medicinal Biotechnology
基金
国家自然科学基金(81001548
30873299
30701123)
上海市教委和上海市教育发展基金会"晨光计划"资助项目(10CG45)
上海市卫生局青年基金(2009Y096)
上海高校优秀青年教师科研基金(szy09022)
上海市重点学科建设项目(S30304)
