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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 15高效表达菌株
Objective To obtain rhIL 15 genetically engineered bacteria. Methods The total cellular RNA was extracted from lung cancer cell line A549 and the fragment encoding the human IL 15 mature region was amplified by RT PCR. After digestion with EcoR I and Xho I, the corresponding restriction enzyme site of the fusion expression plasmid pGEX 4T 2 was inserted into the recombinant fusion protein expression plasmid, and transformed into competent bacteria Escherichia coli BL21. Results The results of the nucleic acid sequence analysis of the recombinant plasmid insert were consistent with the reported nucleic acid sequences of the IL 15 protein mature region. Most of the fusion proteins expressed by this engineering bacterium existed in the form of inclusion bodies, accounting for 39% of the total bacterial proteins. After refolding, the purified rhIL 15 protein was preliminarily determined by MTT colorimetric assay and had the ability to promote CTLL 2 cell proliferation. Conclusion The rhIL 15 highly expressed strains with biological activity were obtained