PDGF-C和TGF-β1的基因克隆原核表达及特异多克隆抗体的制备

发布时间：2018-07-05 10:11

本文选题：血小板衍生因子C + 转化生长因子β1　；参考：《天津医科大学》2008年硕士论文

【摘要】：目的：血小板衍生因子C(PDGF-C)和转化生长因子β1(TGF-B1)是乙型肝炎病毒所导致的肝纤维化的分子病理机制中的重要细胞因子。目前对这两种细胞因子还缺乏有效的检测方法,本研究的目的是克隆人的PDGF-C和TGF-B1的部分编码基因、原核表达蛋白,制备特异性的PDGF-C、TGF-β1多克隆抗体,为建立这两种细胞因子的有效检测方法提供可靠的前提保障。方法：经生物信息学分析,选择出要扩增的人TGF-β1和PDGF-C编码基因片段。通过RT-PCR的方法扩增选择的人TGF-β1和PDGF-C的编码基因,经限制性内切酶EcoRI和Xhol双酶切扩增的目的产物及质粒(pGEX-5X1及pRSET-A2),(?)将经酶切回收的扩增产物及质粒用T4DNA ligase连接酶体外连接组成重组质粒。将重组质粒转化入基因工程克隆菌E.coli XL1-blue进行扩增,将提取的重组质粒进行酶切鉴定及基因测序,明确重组质粒是否正确。将验证正确的重组质粒转化入基因工程表达菌E.coli BL-21或者E.coli BL-21(DE3),在原核细菌中诱导表达出含TGF-β1和PDGF-C部分片段的融合蛋白(带有GST标签),经切胶回收的方法分别纯化出融合蛋白(带有GST标签)。用上述纯化的融合蛋白免疫新西兰大白兔,制备出相应的TGF-β1和PDGF-C多克隆抗体。用含TGF-β1和PDGF-C片段相同,但带HIS标签的融合蛋白,进行ELISA及Western blot检测,并通过检测HepG2细胞内源性表达的PDGF-C和A549细胞中内源性表达的TGF-β1,结合文献报道以明确多克隆抗体是否制备成功。采用Western blot的方式,用制备的抗体检测正常人和肝硬化病人的血清中的PDGF-C和TGF-β1,初步探讨其的应用价值和制备效果。结果：1RT-PCR成功扩增了目的PDGF-C和TGF-β1编码基因片段；2.经基因测序验证,成功构建了pGEX-5X1/PDGF-C重组质粒、pRSET-A2/PDGF-C重组质粒、pGEX-5X1/TGF-β1重组质粒、pRSET-A2/TGF-β1重组质粒；3.转化有重组质粒的E.coli BL-21菌,诱导表达出了条带位置正确的含TGF-β1和PDGF-C部分片段的融合蛋白；4.分别纯化出了TGF-β1、PDGF-C的融合蛋白；5.分别制备了抗TGF-β1、 PDGF-C的兔多克隆抗体；6.使用制备的抗体,对表达的带有HIS标签的PDGF-C、TGF-β1融合蛋白及真核细胞内源性的PDGF-C和TGF-B1蛋白,通过Western blot的方法检测出了目的条带；7.用所制备的抗体进行Western blot检测,发现肝硬化病人血清中的PDGF-C前体和TGF-β1前体含量和正常人有明显差异。结论：成功制备出了特异性的抗人TGF-β1和PDGF-C的兔多克隆抗体,用所制备的抗体对乙肝病毒感染的肝硬化病人和正常人血清中的Western blot检测显示TGF-β1和PDGF-C在肝硬化病人和正常人血清中差异明显,表明制备的抗体对乙型肝炎病毒所导致的肝纤维化的分子病理机制的研究有重要价值,并对建立有效的TGF-β1和PDGF-C的检测方法提供了前提保障。其中对PDGF-C的基因克隆和原核表达及多克隆抗体制备国内外未见他人报道；TGF-β1的基因克隆和原核表达及多克隆抗体制备国内已有人报道,但未见与本文TGF-β1的编码基因部分选取相同的基因克隆和原核表达及多克隆抗体制备。
[Abstract]:Objective: platelet derived factor C (PDGF-C) and transforming growth factor beta 1 (TGF-B1) are important cytokines in the molecular pathological mechanism of liver fibrosis caused by hepatitis B virus. There is still a lack of effective detection methods for these two cytokines. The aim of this study is to clone some of the encoding genes of human PDGF-C and TGF-B1, prokaryotic Expression of the protein, the preparation of specific PDGF-C, TGF- beta 1 polyclonal antibody, for the establishment of effective detection methods for these two cytokines, provide a reliable premise.
Methods: through bioinformatics analysis, the amplified human TGF- beta 1 and PDGF-C encoding gene fragments were selected. The encoding genes of selected human TGF- beta 1 and PDGF-C were amplified by RT-PCR, and the target products and plasmids (pGEX-5X1 and pRSET-A2) were amplified by the restriction endonuclease EcoRI and Xhol, and the amplified products were reclaimed by enzyme digestion. The recombinant plasmid was made up of the T4DNA ligase ligase in vitro. The recombinant plasmid was transformed into the gene engineering clone E.coli XL1-blue for amplification. The recombinant plasmid was identified and the gene was sequenced. The recombinant plasmid was confirmed to be correct. The correct recombinant plasmid was verified to be transformed into the gene engineering expression bacteria E.coli BL-21 or E.coli BL-21 (DE3), the fusion protein containing TGF- beta 1 and PDGF-C fragment (with GST label) was induced in the prokaryotic bacteria. The fusion protein (with GST label) was purified by the method of gel cut recovery. The corresponding TGF- beta 1 and PDGF-C polyclonal antibody was prepared by the purified fusion protein to prepare the corresponding TGF- beta 1 and PDGF-C polyclonal antibody. GF- beta 1 and PDGF-C fragments were the same, but the fusion protein with HIS label was detected by ELISA and Western blot, and the endogenous expression of TGF- beta 1 in PDGF-C and A549 cells expressed in HepG2 cells was detected in the literature to determine whether the polyclonal antibody was prepared successfully. PDGF-C and TGF- beta 1 in serum of normal and cirrhotic patients were preliminarily explored for their application value and preparation effect.
Results: 1RT-PCR amplified target PDGF-C and TGF- beta 1 encoding gene successfully; 2. the recombinant plasmid of pGEX-5X1/PDGF-C, recombinant plasmid of pRSET-A2/PDGF-C, recombinant plasmid of pGEX-5X1/TGF- beta 1, recombinant plasmid of pRSET-A2/TGF- beta 1, and E.coli BL-21 strain of recombinant plasmid were successfully constructed by gene sequencing, and 3. transformed E.coli BL-21 bacteria with recombinant plasmid, and the band position was induced to be expressed. A correct fusion protein containing TGF- beta 1 and PDGF-C partial fragments; 4. purified TGF- beta 1, PDGF-C fusion protein; 5. rabbit polyclonal antibodies against TGF- beta 1, PDGF-C respectively; 6. using the prepared antibodies, HIS labeled PDGF-C, TGF- beta 1 fusion protein and endogenous PDGF-C and TGF-B1 eggs of eukaryotic cells. In white, the target strip was detected by Western blot method; 7. the antibody prepared by the prepared antibody was detected by Western blot, and the contents of PDGF-C precursor and TGF- beta 1 precursor in the serum of patients with liver cirrhosis were significantly different from those of normal people.
Conclusion: the specific anti human TGF- beta 1 and PDGF-C polyclonal rabbit polyclonal antibody was successfully prepared. The detection of Western blot in the serum of liver cirrhosis patients infected by HBV infection and normal human serum showed that the difference of TGF- beta 1 and PDGF-C in the liver cirrhosis patients and normal human serum showed that the prepared antibody was used for hepatitis B disease. The molecular pathological mechanism of liver fibrosis caused by poison is of great value, and provides a prerequisite for the establishment of effective methods for the detection of TGF- beta 1 and PDGF-C. The gene cloning and prokaryotic expression of PDGF-C and the preparation of polyclonal antibodies have not been reported at home and abroad; the gene cloning and prokaryotic expression of TGF- beta 1 and polyclonal resistance to polyclonal resistance are not reported. The preparation has been reported in China, but the same gene clone and prokaryotic expression and polyclonal antibody preparation of the coding gene of TGF- beta 1 have not been found.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R512.62;R3416

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