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目的 :以该室克隆的PCRⅡ hCGβ载体为模板 ,亚克隆到表达载体PG5中使hCGβ基因在大肠杆菌中进行高效表达。方法 :改变部分hCGβ序列 ,得到重组PG5 hCGβ载体 ,经序列分析证明 ,再转化到表达菌BL2 1中表达 ,所得包涵体经过柱纯化 ,复性 ,免疫发光分析和动物试验测定其活性。结果 :经SDS PAGE电泳在 18kD左右有一条明显的新增蛋白带 ,与预期的分子量相符 ,并用Western印迹证实 ,rhCGβ约占菌体总蛋白的 10 % ,活性为 7.8U mg。 结论 :重组hCGβ在大肠杆菌中获得了较高水平的表达。且经复性后有较高的免疫活性和生物活性。
OBJECTIVE: To clone hCGβ gene in E. coli using subcloning PCR Ⅱ hCGβ vector as template and subclone it into expression vector PG5. Methods: The partial hCGβ sequence was changed to obtain the recombinant PG5 hCGβ vector. The recombinant vector was confirmed by sequence analysis and transformed into BL2 1. The inclusion bodies were purified by column, renaturation, immunoluminescence and animal experiments. Results: SDS PAGE showed a significant increase of protein band around 18kD, which was consistent with the expected molecular weight. Western blotting confirmed that rhCGβ accounted for about 10% of the total bacterial protein and the activity was 7.8U mg. Conclusion: Recombinant hCGβ obtained high level of expression in E. coli. And after refolding have higher immunocompetence and biological activity.