摘要
目的研究构建人胱抑素C（Cys C）的重组表达质粒及其在大肠杆菌中的表达和纯化。方法采用反转录-聚合酶链反应从人肝组织中获取Cys C cDNA,并将其插入到pET-28a（＋）质粒中,重组质粒pET-28a-CysC转化入大肠杆菌E.coli BL21（DE3）pLysS,异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达可溶性CysC。重组CysC采用Ni^2＋-NTA系统进行层析纯化。结果核酸序列测定与预期一致,CysC表达水平达到146mg/L,约占菌体总可溶性蛋白的13.5%,表达产物用SDS-PAGE鉴定,其相对分子质量为13.3×10^3,经纯化后蛋白纯度达90%。结论成功构建Cys C原核表达系统和蛋白纯化,为制备抗体以及研制Cys C免疫纳米微球测定试剂奠定了基础。
Objective To study the construction of recombinant cystatin C gene plasmid and its expression in Escherichia coli（E, coli） and protein purification. Methods Cystatin C cDNA was isolated from liver tissue by reverse transcription polymerase chain reaction（RT-PCR）, ligated into pET- 28a（＋） vector, the construct was transformed into E. coli BL21（DE3）pLysS, then the recombinants of cystatin C was induced by IPTG, purified by affinity chromatography through Ni^2＋-NTA. Results The acquired gene was 380 bp and its sequence was correct. The construct was expressed in E. coli with a high level as a soluble protein,accounting for 13.5% of the total batterial proteins. The gene product, characterized by SDS-PAGE appeared to be a protein with molecular mass of 13 300. The purity of the protein purified by affinity chromatography reached more than 90%0. Conclusion The success of construction,expression and purification of human cystatin C in E. coli provides basis for studying the preparation of polyclonal antibody and diagnostic reagent.
出处
《国际检验医学杂志》
CAS
2007年第5期393-394,共2页
International Journal of Laboratory Medicine
