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目的 :人血小板生成素 (TPO)在大肠杆菌中的高效表达。方法 :利用PCR技术 ,扩增出了( 1～ 174个氨基酸的 )人血小板生成素cDNA ,并将其克隆到pGEX 1λT载体中 ,构建了融合蛋白表达质粒。重组子用限制性内切酶酶切鉴定 ,表达产物经SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)。结果 :限制性内切酶酶切鉴定表明重组子含有正确的 ( 1～ 174氨基酸 )TPOcDNA片段 ;表达产物经SDS PAGE相对分子质量约为 4 30 0 0 ,与预期的相符合 ,表达占可溶性菌体总蛋白的 4 4 .56%。结论 :表达产物具有明显的刺激Mo7e细胞增殖与CFU Meg生成的功能。
OBJECTIVE: Highly efficient expression of human thrombopoietin (TPO) in Escherichia coli. METHODS: Human thrombopoietin cDNA (1 to 174 amino acids) was amplified by PCR and cloned into pGEX 1λT vector to construct a fusion protein expression plasmid. The recombinants were identified by restriction enzyme digestion and expressed by SDS polyacrylamide gel electrophoresis (SDS PAGE). Results: Restriction endonuclease digestion indicated that the recombinant contained the correct (1 to 174 amino acids) TPO cDNA fragments. The SDS PAGE molecular weight of the expressed product was about 4 30 0 0, which was consistent with the expectation. 44.56% of the total body protein. Conclusion: The expressed product has the obvious function of stimulating the proliferation of Mo7e cells and CFU Meg generation.