摘要
目的 获得rhIL 15基因工程菌。方法 从肺癌细胞株A549中提取细胞总RNA ,用RT PCR法扩增编码人IL 15成熟区基因的片段。经EcoRⅠ和XhoⅠ双酶切后 ,插入融合表达质粒pGEX 4T 2的相应酶切位点 ,构建重组融合蛋白表达质粒 ,并转化感受态大肠杆菌BL2 1而得到工程菌。结果 重组质粒插入片段核酸序列测定的结果 ,与文献报道的IL 15蛋白成熟区的核酸序列相一致。该工程菌所表达的融合蛋白多数以包涵体的形式存在 ,占菌体蛋白总量的 39%。复性后 ,纯化的rhIL 15蛋白经MTT比色法初步测定 ,具有促进CTLL 2细胞增殖的能力。结论 获得了具有生物学活性的rhIL
Aim To obtain rhIL 15 expression E.coli . Methods The cDNA fragment coding for mature IL 15 protein was amplified by RT PCR from lung carcinoma cell line A 549 . The gene fragment was cloned into fusion expression vector pGEX 4T 2 digested with Eco RⅠ and Xho Ⅰ. The recombinant plasmid was transformed into E.coli BL21. Results Sequencing result showed that the cloned fragment of hIL 15 gene was identical with that having been reported. The fusion protein of GST IL 15 was expressed mainly in the form of inclusion bodies, accounting for more than 39% of the total bacterial protein. After purification and renaturation, the fusion protein could stimulate the proliferation of T cell line CTLL 2. Conclusion E.coli that efficiently express rhIL 15 with biological activity has been obtained.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第4期332-334,338,共4页
Chinese Journal of Cellular and Molecular Immunology
