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建立人FL(humanflt3ligand ,hFL)在大肠杆菌中的高效表达系统 ,分离纯化重组hFL为其功能和应用研究打下基础。根据大肠杆菌偏爱密码子人工合成hFL基因膜外区DNA片段 ,由PCR方法获得的hFL基因膜外区DNA片段 ,经测序证明序列正确。构建表达载体pET30a Trx hFL并转化大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,hFL融合蛋白的表达占菌体总蛋白的 6 0 %以上 ,并以包涵体形式存在。融合蛋白经离子交换层析、分子筛层析纯化 ,并用FXa切割 ,切割效率 >80 %。切割产物经亲和层析纯化。纯化产物进行Westernblot鉴定。纯化的rhFL(recombinanthFL)与GM CSF及TNFα联合作用 ,具有良好的刺激DC增殖活性 ,其增殖能力是GM CSF +TNFα的 2 5倍左右。本文以大肠杆菌为宿主 ,成功地表达了融合蛋白Trx hFL。经纯化的rhFL在体外与GM CSF及TNFα联合使用能有效地刺激人DC的增殖。
The efficient expression system of human FL (human FLT3ligand, hFL) in E.coli was established, and the isolation and purification of recombinant hFL laid the foundation for its function and application. According to the codon preference of E. coli, the hFL gene extracellular DNA fragment was synthesized by PCR. The DNA fragment of hFL gene extracellular region obtained by PCR method was verified by sequencing. The expression vector pET30a Trx hFL was constructed and transformed into E. coli BL21 (DE3). The expression of hFL fusion protein was induced by IPTG. The expression of hFL fusion protein accounted for more than 60% of total bacterial proteins, and existed as inclusion body. The fusion protein was purified by ion exchange chromatography and molecular sieve chromatography and cleaved with FXa. The cleavage efficiency was> 80%. The cleavage product was purified by affinity chromatography. The purified product was identified by Western blot. Purified rhFL (recombinant hFL) and GM CSF and TNFα combined with a good stimulating DC proliferative activity, the proliferation of GM CSF + TNFα 25 times. In this paper, Escherichia coli as a host, the successful expression of the fusion protein Trx hFL. Purified rhFL in vitro and GM CSF and TNFα in combination can effectively stimulate the proliferation of human DC.