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利用酵母真核细胞表达系统表达出重组人内皮抑制素 (Endostatin) ,并对其促成纤维细胞有丝分裂活性进行研究。方法 :采用逆转录 PCR技术自人肝组织获得人内皮抑制素的cDNA ,将其克隆入真核表达载体pYEX4T 1,构建含人Endostatin的重组质粒 (pYEXEndo) ;经全自动序列分析仪测序确证后 ,将此重组质粒转化入酵母细胞 (DY15 0 )中进行诱导表达 ,表达产物经SDS PAGE分析及Westernblot鉴定。以MTT掺入法初步检测了重组人内皮抑制素对NIH3T3细胞的促有丝分裂活性。结果 :经CuSO4诱导的含pYEXEndo的酵母细胞表达出重组人GST Endostatin的融合蛋白 ,此蛋白在凝胶上表现为一约 45kD的阳性区带 ,在Westernblot实验中可被GST特异性多克隆抗体识别。重组人GST Endostatin融合蛋白的粗提物在体外能刺激NIH3T3细胞增殖。结论 :人GST Endostatin的融合蛋白在酵母表达系统中高水平表达 ,并具有刺激成纤维细胞生长的生物学活性。
Recombinant human endostatin was expressed in yeast eukaryotic expression system and its mitogenic activity was studied. METHODS: cDNA of human endostatin was obtained from human liver tissue by RT-PCR and cloned into the eukaryotic expression vector pYEX4T 1 to construct the recombinant plasmid pYEXEndo containing endostatin. After sequencing by automatic sequence analyzer The recombinant plasmids were transformed into yeast cells (DY15 0) to induce expression. The expressed products were identified by SDS PAGE and Western blot. The MTT assay was used to detect the mitogenic activity of recombinant human endostatin on NIH3T3 cells. Results: The fusion protein of recombinant human GST Endostatin was expressed in CuSO4-containing pYEXEndo-containing yeast cells. The protein showed a positive band of about 45 kD on the gel and was recognized by GST-specific polyclonal antibody in Western blotting . Crude extracts of recombinant human GST Endostatin fusion protein stimulate NIH3T3 cell proliferation in vitro. CONCLUSION: The fusion protein of human GST Endostatin is highly expressed in yeast expression system and has the biological activity of stimulating the growth of fibroblasts.