心肌型脂肪酸結(jié)合蛋白原核表達載體（pBV）的構(gòu)建、表達及純化
發(fā)布時間：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載體pBV-220重組,得到了pBV-H-FABP重組表達載體;成功地將重組質(zhì)粒轉(zhuǎn)化到大腸桿菌DH5α中,通過溫度誘導表達出了成熟的人心肌型脂肪酸結(jié)合蛋白;通過反復凍融,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重組表達是目前獲得H-FABP蛋白含量最高的基因工程表達方法。 H-FABP的成功表達為進一步研究H-FABP的功能及制備單克隆抗體奠定了基礎。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346
【參考文獻】
相關期刊論文 前6條
1 張智清,姚立紅,侯云德;含P_RP_L啟動子的原核高效表達載體的組建及其應用[J];病毒學報;1990年02期
2 楊文東,馬慶海;病毒性心肌炎患兒治療前后血清脂肪酸結(jié)合蛋白水平變化及臨床意義[J];濱州醫(yī)學院學報;2002年01期
3 ;Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris[J];Chemical Research in Chinese Universities;2006年02期
4 楊振華;生化標志物在缺血性心臟病診斷中的臨床價值[J];中華醫(yī)學檢驗雜志;1997年06期
5 王顯花,崔紅蓮,孫冬梅,胡美浩,衣志勇,李小鷹;心臟型脂肪酸結(jié)合蛋白診斷急性心肌梗死早期指標的可行性研究[J];中國老年學雜志;2003年03期
6 侯巍,許淑芬,劉玲麗,楊曉虹,譚巖;人心肌型脂肪酸結(jié)合蛋白(H-FABP)在大腸桿菌中的表達和純化[J];中國實驗診斷學;2005年03期
，本文編號：2250898
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