心肌型脂肪酸結(jié)合蛋白原核表達(dá)載體（pBV）的構(gòu)建、表達(dá)及純化
發(fā)布時(shí)間：2018-09-19 17:58
【摘要】：本研究從人胚胎心肌組織提取總RNA,通過逆轉(zhuǎn)錄PCR擴(kuò)增人心肌型脂肪酸結(jié)合蛋白的基因片斷(396bp),首次將目的基因與原核表達(dá)載體pBV-220重組,得到了pBV-H-FABP重組表達(dá)載體;成功地將重組質(zhì)粒轉(zhuǎn)化到大腸桿菌DH5α中,通過溫度誘導(dǎo)表達(dá)出了成熟的人心肌型脂肪酸結(jié)合蛋白;通過反復(fù)凍融,使菌體上清中含有較多的可溶性脂肪酸結(jié)合蛋白,減少了分離包涵體及包涵體變性、復(fù)性的過程。菌體上清先后經(jīng)SephacrylS-100 HR柱和Sepharose G-75柱純化后得到重組H-FABP,含量約為850mg·L-1,利用pBV-220重組表達(dá)是目前獲得H-FABP蛋白含量最高的基因工程表達(dá)方法。 H-FABP的成功表達(dá)為進(jìn)一步研究H-FABP的功能及制備單克隆抗體奠定了基礎(chǔ)。
[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 偽, 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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
本文編號：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 偽, 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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346
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，本文編號：2250898
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