重组人糖皮质激素受体原核表达载体的构建和表达

发布时间：2018-04-29 07:54

本文选题：阴阳虚证 + 糖皮质激素受体　；参考：《第二军医大学》2005年硕士论文

【摘要】：目的:构建hGRα重组原核表达质粒并进行诱导表达,为下一步研制hGRα ELISA检测试剂盒和进一步研究GR与中医学阴阳虚证的关系服务。方法:以含有hGRα cDNA的重组克隆质粒pRShGRα为模板,通过PCR扩增得到hGRα的两种基因片段,将这两种基因片段分别克隆到pGEM-T载体上,进而亚克隆到pGEX-4T-3表达载体上。将这两种重组表达质粒进行酶切、测序鉴定后,分别转化大肠杆菌DH5α和BL21(DE3),并在一定的条件下进行重组蛋白的诱导表达,诱导表达产物行SDS-PAGE和Western blotting分析。结果:酶切和测序结果证明hGRα的两种基因片段均成功地重组入pGEX-4T-3载体。诱导表达产物的SDS-PAGE和Western blotting分析均提示有hGRα目的蛋白特异表达。结论:本实验成功构建了hGRα的两种重组原核表达质粒,并成功诱导了它们的原核表达,为下一阶段的研究奠定了基础。
[Abstract]:Objective: to construct the recombinant prokaryotic expression plasmid of hGR 伪 and express it in order to further study the relationship between gr and yin and yang deficiency syndrome in traditional Chinese medicine. Methods: using the recombinant clone plasmid pRShGR 伪 containing hGR 伪 cDNA as template, two kinds of hGR 伪 gene fragments were amplified by PCR and cloned into pGEM-T vector respectively, and then subcloned into pGEX-4T-3 expression vector. The two recombinant expression plasmids were digested by enzyme and identified by sequencing. The recombinant proteins were transformed into Escherichia coli DH5 伪 and BL21DE-3, respectively. The recombinant protein was induced and expressed under certain conditions. The induced expression products were analyzed by SDS-PAGE and Western blotting. Results: the results of restriction endonuclease digestion and sequencing showed that the two gene fragments of hGR 伪 were successfully recombined into pGEX-4T-3 vector. Both SDS-PAGE and Western blotting analysis of induced expression products indicated that hGR 伪 target protein was specifically expressed. Conclusion: in this experiment, two recombinant prokaryotic expression plasmids of hGR 伪 were successfully constructed, and their prokaryotic expression was induced successfully, which laid a foundation for the next stage of research.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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