心肌型脂肪酸结合蛋白原核表达载体（pBV）的构建、表达及纯化
发布时间：2018-09-19 17:58
【摘要】：本研究从人胚胎心肌组织提取总RNA,通过逆转录PCR扩增人心肌型脂肪酸结合蛋白的基因片断(396bp),首次将目的基因与原核表达载体pBV-220重组,得到了pBV-H-FABP重组表达载体;成功地将重组质粒转化到大肠杆菌DH5α中,通过温度诱导表达出了成熟的人心肌型脂肪酸结合蛋白;通过反复冻融,使菌体上清中含有较多的可溶性脂肪酸结合蛋白,减少了分离包涵体及包涵体变性、复性的过程。菌体上清先后经SephacrylS-100 HR柱和Sepharose G-75柱纯化后得到重组H-FABP,含量约为850mg·L-1,利用pBV-220重组表达是目前获得H-FABP蛋白含量最高的基因工程表达方法。 H-FABP的成功表达为进一步研究H-FABP的功能及制备单克隆抗体奠定了基础。
[Abstract]:In this study, total RNA, was extracted from human embryonic myocardium to amplify the gene fragment of human myocardial fatty acid binding protein (396bp) by reverse transcription PCR. The target gene was recombined with prokaryotic expression vector pBV-220 for the first time, and the recombinant expression vector of pBV-H-FABP was obtained. The recombinant plasmid was successfully transformed into Escherichia coli DH5 伪, and the mature human myocardial fatty acid binding protein was expressed by temperature induction, and the supernatant contained more soluble fatty acid binding proteins through repeated freezing and thawing. The separation of inclusion bodies and the process of renaturation and renaturation of inclusion bodies are reduced. After purified by SephacrylS-100 HR column and Sepharose G-75 column, the recombinant H-FABP was obtained with the content of 850mg L-1.Using pBV-220 recombinant expression was the highest expression method of H-FABP protein at present. The successful expression of H-FABP lays a foundation for the further study of the function of H-FABP and the preparation of monoclonal antibodies.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：2250898
[Abstract]:In this study, total RNA, was extracted from human embryonic myocardium to amplify the gene fragment of human myocardial fatty acid binding protein (396bp) by reverse transcription PCR. The target gene was recombined with prokaryotic expression vector pBV-220 for the first time, and the recombinant expression vector of pBV-H-FABP was obtained. The recombinant plasmid was successfully transformed into Escherichia coli DH5 伪, and the mature human myocardial fatty acid binding protein was expressed by temperature induction, and the supernatant contained more soluble fatty acid binding proteins through repeated freezing and thawing. The separation of inclusion bodies and the process of renaturation and renaturation of inclusion bodies are reduced. After purified by SephacrylS-100 HR column and Sepharose G-75 column, the recombinant H-FABP was obtained with the content of 850mg L-1.Using pBV-220 recombinant expression was the highest expression method of H-FABP protein at present. The successful expression of H-FABP lays a foundation for the further study of the function of H-FABP and the preparation of monoclonal antibodies.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
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