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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 fetal lung was taken as primary cultured cells. After induced by PMA, IL 11 cDNA with N-terminal signal peptide sequence was amplified by RT-PCR. Sequence analysis showed that the sequence was highly homologous to the reported sequence A nucleotide changes, the amino acid sequence is exactly the same. The IL 11 cDNA was cloned into the 3 ’end of the trxA gene of the thioredoxin gene fusion expression vector pTRXFUS, and the 3’ end protein enterokinase cut point was used to construct a frame-like fusion gene. The fusion protein expressed more than 20% in E. coli. The biological activity was determined by IL-6-dependent cell line 7TD1 and MTT assay, reaching 256 × 105u / mL. The fusion protein was determined by Western blot and the expression conditions were preliminarily studied.