摘要
目的 构建人CD2 2 6 (PTA1)分子胞膜外区的原核表达载体 ,并进行原核表达和复性 ,为该分子的X线结晶衍射及功能学研究提供充足的蛋白来源。方法 将人CD2 2 6分子的胞膜外区基因克隆入原核表达载体pBV2 2 0 ,测序证实后 ,转化宿主菌MC10 6 1 P3并进行温度诱导表达。产物采用稀释加透析法复性 ,亲和层析柱纯化 ,并通过Westernblot和ELISA对其复性效果进行鉴定。结果 温度诱导 7h ,目的蛋白的表达量达宿主菌总蛋白量的 2 3.8% ,其相对分子质量为 30× 10 3,Westernblot及ELISA证实其重折叠和复性是成功的。结论 成功地获得了人CD2 2 6 (PTA1)分子胞膜外区的原核表达产物 。
Objective To construct the prokaryotic expression vector and express human CD226 (PTA1), in order to obtain CD226 recombinant molecules for X ray crystallographic analysis and functional study. Methods The gene fragment encoding extracellular region of human CD226 was cloned into a prokaryotic expression vector pBV220. After sequencing, the vector was transferred into MC1061/P3 and the expression of the molecule was induced. Finally, the product was renatured by dilution and dialysis, and then purified by affinity chromatography. The products were characterized by Western blot and ELISA. Relsults After 7 hours of temperature induction, the expressed protein with molecule weight of 30kD was amount to 23.8% of the total protein of the host cell. The refolding renaturation was successful confirmed by Western blot and ELISA. Conclusion The prokaryotic expression of the extracellular region of human CD226 is successful, which lays a foundation for the X ray crystallographic analysis and functional study of this molecule.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2002年第6期587-590,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金重点项目(3 0 0 3 0 13 0 )
NationalKeyBasicResearchProgramofChina(20 0 1CB5 10 0 0 4)
面上项目(3 9980 0 0 6)
