重组人突变型471TNF-α及野生型TNF-α的表达及活性初步鉴定

The expression of recombinant human mutant 471 TNF-α and with type TNF-α and the primary identification of their bioactivities

目的 采用原核表达系统 ,表达人突变型 471TNF α蛋白 ,并检测其生物学活性。方法 采用PCR技术扩增突变型 471TNF α及野生型TNF αcDNA片段 ,克隆入中间载体pUCm T中 ,并测序证实。以限制性内切酶切下目的片段 ,克隆入pBV2 2 0中 ,转入大肠杆菌DH5α ,温度诱导表达野生型及突变型 471TNF α蛋白。初步纯化蛋白后 ,采用MTT法评估两者对L92 9细胞的杀伤作用。结果 ①获得了测序正确的野生型TNF α及突变型 471TNF α的cDNA克隆 ;②构建了正常人野生型TNF α及突变型 471TNF α重组表达质粒 ;③经温度诱导 ,TNF α及突变型 471TNF α蛋白均获得了表达 ;④初步的生物学活性研究表明 ,突变型 471TNF α蛋白的杀伤作用显著高于野生型。结论 突变型 471TNF α的生物学活性优于野生型TNF α ,为进一步开展TNF

Objective To construct and express the mutant 471 rhTNF-α with prokaryotic expression system and to compare the differences between the mutant 471 and wild type rhTNF-α bioactivities. Methods The PCR fragments of human wild type and mutant 471 rhTNF-α were cloned into the mediate vector pUCm-T. After sequencing and digesting, the inserted target fragments were then cloned into prokaryotic expression system pBV220 and E.coli DH5α was transformed as a competent cell with plasmids. The expressed proteins of mutant 471 and wild type TNF-α under the inducing of temperature were primarily purified and their biological activities were evaluated by MTT method with L929 cells as target cells. Results We successfully constructed the recombinant cloning and expressing plasmids of human wild type and mutant 471 rhTNF-α and obtained the human wild type and mutant 471 TNF-α proteins from E.coli DH5α expressin system under the inducing of temperature. Mutant 471 and had a markedly higher cytotoxic activity against murine L929 cells than wild type rhTNF-α. Conclusion The biological activity of mutant 471 rhTNF-α protein is superior to that of wild type rhTNF-α, which lays a foundation for further basic and clinical studies.