摘要
目的 构建含有人THANK基因的表达载体 ,诱导其在大肠杆菌中可溶性表达 ,并对表达的THANK蛋白的免疫调节活性进行检测。方法 从含有全长THANKcDNA的pMD18 THANK质粒中克隆THANK的胞外区片段 ,并将其亚克隆至原核表达载体pET 11a中 ,筛选阳性重组质粒pET THANK ,以IPTG诱导其可溶性表达 ,并以SDS PAGE和Westernblot检测进行分析。表达的蛋白初步纯化后 ,分析其对B、T细胞的免疫调节活性。结果 PCR扩增出了THANK胞外区cDNA片段。SDS PAGE和Western分析证实重组的pET THANK质粒可表达出THANK蛋白。可溶性THANK重组蛋白可共刺激B、T细胞的增生 ,并可诱导活化T细胞的凋亡。结论 本实验成功地将THANK胞外区片段在大肠杆菌中进行表达 。
Objective To construct THANK expressing vector and induce its protein expression in E.coli and to analyze its bioactivity. Methods The extracellular fragment of THANK was amplified by PCR using pMD18 THANK plasmid containing a full length THANK cDNA and the extracellular fragment was subcloned into the pET 11a vector resulting in a recombinant plasmid pET THANK, the plasmid was inducted with IPTG in different conditions and the expressed protein was analyzed and identified by SDS PAGE/Western blot. The function of purified THANK protein was identified. Results THANK extracellular cDNA was amplified by PCR, SDS PAGE. Western blot analysis showed that THANK protein was expressed. Further bioactivity assay indicated that the soluble protein can costimulate B, T cell proliferation and induced activated T cells apoptosis. Conclusion THANK extracellular cDNA was successfully cloned and expressed. The bioactivity analysis showed that the protein was functioning in the modulation of immune response.
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603-607,共5页
Chinese Journal of Microbiology and Immunology
