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目的: 克隆人tumstatin全长编码区基因及编码tum statin45-132的基因, 在大肠杆菌中表达。方法: 采用RT PCR从人胚肾细胞系 293细胞中克隆人的tumstatin全长编码基因, 继以PCR扩增tumstatin45-132 (45 -132位氨基酸 )编码基因, PCR产物克隆到pBV220载体中, 测序证实后, 转化E.coliBL21, 于 42℃进行热诱导表达。用SDS -PAGE分析表达产物, 对重组蛋白纯化后用内皮细胞增殖试验测定其生物学活性。结果: RT- PCR扩增出tumstatin全长编码基因, 以PCR扩增出tumstatin45-132的编码基因, 经序列分析证实与GenBank中的序列完全一致。以含有tumstatin45-132编码基因的表达载体转化E.coliBL21后, 可表达出相对分子质量(Mr)为 9 600的重组蛋白。表达产物的蛋白量占菌体蛋白量的 10%, 纯化后能抑制内皮细胞的增殖。结论: 成功克隆全长tumstatincDNA, 并在大肠杆菌中表达重组tumstatin45-132蛋白, 证实其具有抑制内皮细胞增殖的活性。
Objective: To clone the full length coding region of human tumstatin gene and the gene encoding tum statin45-132 and express in E. coli. METHODS: Human tumstatin full-length coding gene was cloned by RT-PCR from 293 human embryo kidney cell line. The gene encoding tumstatin45-132 (amino acids 45-132) was amplified by PCR. The PCR product was cloned into pBV220 vector and sequenced After confirmation, E. coli BL21 was transformed and induced by heat at 42 ° C. The expression product was analyzed by SDS-PAGE, and the biological activity of the recombinant protein was measured by the endothelial cell proliferation assay after purification. Results: The full-length tumstatin gene was amplified by RT-PCR, and the gene encoding tumstatin45-132 was amplified by PCR. The sequence of tumstatin45-132 was confirmed by sequence analysis. After transforming E. coli BL21 with the expression vector containing the gene encoding tumstatin45-132, a recombinant protein with molecular weight (Mr) of 9 600 can be expressed. The expression product of protein accounted for 10% of the amount of bacterial protein, purified can inhibit the proliferation of endothelial cells. Conclusion: The full length tumstatincDNA was successfully cloned and the recombinant tumstatin45-132 protein was expressed in E. coli, which confirmed that it has the activity of inhibiting the proliferation of endothelial cells.