sTRAIL基因的表达、纯化及其生物学活性的初步研究

在原核表达系统中表达重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(sTRAIL)分子，纯化表达产物，并进行初步的生物学活性的研究中，通过提取外周血淋巴细胞总RNA进行RT—PCR克隆sTRAIL cDNA，构建sTRAIL的原核表达载体，用限制性酶切和DNA测序进行鉴定，IPTG诱导表达。以发酵罐放大培养，SDS-PAGE和Western-blot确认表达，用亲和层析和阳离子层析纯化表达产物，使用L929、Qay肝癌、K562人白血病等肿瘤细胞鉴定活性。结果发现重组表达的sTRAIL融合蛋白占总蛋白的39％，单纯蛋白纯化后纯度达95％，用抗人TRAIL多抗可以确认表达了TRAIL分子，能诱导几株肿瘤细胞凋亡。原核表达系统正确表达了TRAIL分子，并摸索了中试生产的条件，为其在肿瘤生物治疗中的应用奠定了基础。

Expression , purification and investigation of sTRAIL

To investigate the soluble part of tumor necrosis factor related apoptosis inducing ligand (TRAIL)expression in E.coli,purify the expressed product and examine its biological characters.RT PCR was used to preparation of the soluble part of TRAIL,which is encoded 95 281 amino acid.The molecule used in the clone process was conformed by restrictive enzyme digestion and sequencing.By IPTG induction,the expressed product was identified by SDS PAGE and Western blot.The expression was magnified by fermentor and purified by affinity and cation chromatograph.The cell lines L929 Qay and K562 were used to detect of cytotoxicity of the product.SDS PAGE analysis indicated that a new fusion protein band was found corresponding at 34KD,which made up 39% of the total proteins.The purity of simple protein is 95%.The anti TRAIL polyclonal antibody could combine with the expressed protein from E.coli. The cytotoxicity was observed to cell lines L929 Qay and K562.The soluble part of TRAIL was correctly exp essed in E.coli and the attempt tried to find the suitable conditions in middle scale production,which will provide fundamental study in pathological roles of TRAIL mediated cytotoxicity and its potential application to tumor immunotherapy.