人白细胞介素11cDNA的克隆、融合表达及活性测定  

cDNA Cloning,Fusion Expression in Escherichia coli and Activity Assay of hIL-11

作　　者：张颖[1] 刘灿辉[1] 刘玉乐[1] 田波[1] 

机构地区：[1]中国科学院微生物研究所,北京100080

出　　处：《生物工程学报》2000年第3期353-356,共4页Chinese Journal of Biotechnology

摘　　要：取人胎肺做原代培养细胞 ,PMA诱导后 ,利用RT PCR技术 ,扩增出了去掉N端信号肽序列的IL 11cDNA ;经序列分析表明 ,该序列与文献报道序列高度同源 ,仅 3个核苷酸有变化 ,氨基酸序列完全一致。将此IL 11cDNA克隆入硫氧还蛋白基因融合表达载体 pTRXFUS的trxA基因 3′末端 ,利用其 3′端的蛋白肠激酶切点 ,构建符合读码框的融合基因。该融合蛋白在大肠杆菌中表达量达 2 0 %以上。用IL 6依赖细胞株 7TD1及MTT法测定生物学活性 ,达 2 5 6× 10 5u/mL菌液 ,对融合蛋白进行了Westernblot测定 ,并对表达条件作了初步研究。Human Interleukin\|11 (hIL\|11) is a multifunctional cytokine which plays an important role in regulating the proliferation and differentiation of cells in the hematopoeitic,lymphoid system etc. To obtain the IL\|11 cDNA,a primary culture of Chinese fetal lung fibroblast was prepared from fresh tissue.Then the human IL\|11 cDNA without the N\|terminal signal peptide sequence was cloned by RT\|PCR from the cells induced by PMA.The sequnce indicated that there are three bases different from those previously reported,but with no change of the amino acids.The cDNA was inserted into the 3′ end of trxA gene in thioredoxin gene fusion expression system pTRXFUS to construct the trxA and hIL\|11 fusion expression vector,and expressed in E.coli. The fusion hIL\|11 accounts for more than 20% of the total bacteria proteins.The expression product is present in soluble forms and has the full biological and immunological activities.

关 键 词：人白细胞介素11 活性测定 基因克隆 融合表达 

分 类 号：Q753[生物学—分子生物学]