【作者】 巩宏伟；

【导师】 杨晓虹；

【作者基本信息】 吉林大学 ， 药物化学， 2006， 硕士

【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。更多还原

【Abstract】 Fatty acid-binding proteins (FABPs) are a member of the conservativemulti-gene family of in-cell lipid-binding proteins(iLBPs). They were firstdiscovered on rat intestinal mucous by Ockner in 1972 when he was engagedin the research of the modulation of absorbing the fatty acid in small intestineof the rat. FABPs are expressed in many kinds of mammal cells, such as smallintestine, liver, fat, heart, brain and skeletal muscle, etc and their relative MWare approximately 12-16kD. FABPs were designated by their distribution inthe tissues, and nine types of FABPs are identified to date, including:I-FABP(intestinal fatty acid-binding protein), H-FABP(heart type fattyacid-binding protein), L-FABP(liver fatty acid-binding protein),K-FABP(kidney fatty acid-binding protein) etc. Several kinds of FABPs can beexpressed in one cell at the same time , for example , there are two differentFABPs , L-FABP and I-FABP, on the intestine endothelial cells . Thesequences of different types of FABPs have more homology.Heart-type fatty acid-binding protein (H-FABP) is a kind of dissolvablecytoplasm protein and its relative MW is approximately 14-15KD. It is anacidic protein and the isoelectric point(pl) is 5.1, The primary structure ofhuman?s H-FABP is composed of 132 amino acids , in which there are plentyof threonines and lysines, but deficiency of cysteine, and with a residue ofacetyl valine on its N terminal.H-FABP has the specific binding ability with hydrophobic ligandmolecules, such as LCFA(long-chain fatty acid), vitamin(vitamin A1),RA(retinoic acid) and some organic negative ions. H-FABP is the key carrierprotein of fatty acid. It can transport the fatty acid from cytomembrane to theparts of esterization and oxidation and thereby enter the energy metabolismsystem of mitochondria where the fatty acid are oxided and broken down,producing ATP to provide energy.H-FABP particularly exists in cardiac muscle in large amount, about 4%-8% of the dissolvable proteins of the whole heart. For the human adult, eachgram of cardiac muscle contains about 0.5mg of H-FABP . There is no detect-able H-FABP or just trace amount in the blood plasma and urine of health adu-lt. Because of its low molecular weight and cytoplasmic location, H-FABP israpidly released into the circulation after myocardial injury. H-FABP will app-ear in the blood during the 1.5hours after the acute myocardial infarction, andthe content in the blood will reach the peak during the first 4-6 hours, then ret-urn to the baseline after 24 hours. Thereby, it is an ideal marker for the earlydiagnosis of acute myocardial infarction. Moreover, it can also be helpful tothe judgment of treatment efficacy and prognosis . In addition, H-FABP canalso be taken as a marker to detect the recurrence of acute myocardial infarct-tion and evaluate the infarct size.In our research, the total RNA was isolated from cardiac muscle ofhuman fetus. RT-PCR technique was used to amplify the gene fragments ofhuman H-FABP(396bp). Then the target gene was cloned into the prokaryoticexpression vector pBV220 to construct recombinant expressing plasmidpBV-H-FABP for the first time. The recombinant plasmid pBV-H-FABP weretransformed into E.coli DH5α and successfully expressed human fattyacid-binding protein by temperature inducement. We obtained lots ofdissolvable fatty acid-binding protein in the supernatant by freezing-thawingof the transformed E.coli. This escaped the process of separation, degenerationand comebacking renaturation of inclusion body. This is a innovation in ourresearch. We got the purified recombinant H-FABP, and the concentration is850mg·L-1, by making the supernatant to pass through the columns ofSephacryl S-100 HR and Sepharose G-75 successively. The gene engineeringexpression methods by pBV-220 vector is the best one to get the highestconcentration of recombinant H-FABP to data.The successful expression of H-FABP lay a preliminary basis for furtherinvestigation of the function of H-FABP and the preparation of monoclonalantibodies against H-FABP.更多还原