發(fā)布時(shí)間：2018-07-05 10:11

  本文選題：血小板衍生因子C + 轉(zhuǎn)化生長因子β1��； 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】：目的：血小板衍生因子C(PDGF-C)和轉(zhuǎn)化生長因子β1(TGF-B1)是乙型肝炎病毒所導(dǎo)致的肝纖維化的分子病理機(jī)制中的重要細(xì)胞因子。目前對這兩種細(xì)胞因子還缺乏有效的檢測方法,本研究的目的是克隆人的PDGF-C和TGF-B1的部分編碼基因、原核表達(dá)蛋白,制備特異性的PDGF-C、TGF-β1多克隆抗體,為建立這兩種細(xì)胞因子的有效檢測方法提供可靠的前提保障。 方法：經(jīng)生物信息學(xué)分析,選擇出要擴(kuò)增的人TGF-β1和PDGF-C編碼基因片段。通過RT-PCR的方法擴(kuò)增選擇的人TGF-β1和PDGF-C的編碼基因,經(jīng)限制性內(nèi)切酶EcoRI和Xhol雙酶切擴(kuò)增的目的產(chǎn)物及質(zhì)粒(pGEX-5X1及pRSET-A2),(?)將經(jīng)酶切回收的擴(kuò)增產(chǎn)物及質(zhì)粒用T4DNA ligase連接酶體外連接組成重組質(zhì)粒。將重組質(zhì)粒轉(zhuǎn)化入基因工程克隆菌E.coli XL1-blue進(jìn)行擴(kuò)增,將提取的重組質(zhì)粒進(jìn)行酶切鑒定及基因測序,明確重組質(zhì)粒是否正確。將驗(yàn)證正確的重組質(zhì)粒轉(zhuǎn)化入基因工程表達(dá)菌E.coli BL-21或者E.coli BL-21(DE3),在原核細(xì)菌中誘導(dǎo)表達(dá)出含TGF-β1和PDGF-C部分片段的融合蛋白(帶有GST標(biāo)簽),經(jīng)切膠回收的方法分別純化出融合蛋白(帶有GST標(biāo)簽)。用上述純化的融合蛋白免疫新西蘭大白兔,制備出相應(yīng)的TGF-β1和PDGF-C多克隆抗體。用含TGF-β1和PDGF-C片段相同,但帶HIS標(biāo)簽的融合蛋白,進(jìn)行ELISA及Western blot檢測,并通過檢測HepG2細(xì)胞內(nèi)源性表達(dá)的PDGF-C和A549細(xì)胞中內(nèi)源性表達(dá)的TGF-β1,結(jié)合文獻(xiàn)報(bào)道以明確多克隆抗體是否制備成功。采用Western blot的方式,用制備的抗體檢測正常人和肝硬化病人的血清中的PDGF-C和TGF-β1,初步探討其的應(yīng)用價(jià)值和制備效果。 結(jié)果：1RT-PCR成功擴(kuò)增了目的PDGF-C和TGF-β1編碼基因片段；2.經(jīng)基因測序驗(yàn)證,成功構(gòu)建了pGEX-5X1/PDGF-C重組質(zhì)粒、pRSET-A2/PDGF-C重組質(zhì)粒、pGEX-5X1/TGF-β1重組質(zhì)粒、pRSET-A2/TGF-β1重組質(zhì)粒；3.轉(zhuǎn)化有重組質(zhì)粒的E.coli BL-21菌,誘導(dǎo)表達(dá)出了條帶位置正確的含TGF-β1和PDGF-C部分片段的融合蛋白；4.分別純化出了TGF-β1、PDGF-C的融合蛋白；5.分別制備了抗TGF-β1、 PDGF-C的兔多克隆抗體；6.使用制備的抗體,對表達(dá)的帶有HIS標(biāo)簽的PDGF-C、TGF-β1融合蛋白及真核細(xì)胞內(nèi)源性的PDGF-C和TGF-B1蛋白,通過Western blot的方法檢測出了目的條帶；7.用所制備的抗體進(jìn)行Western blot檢測,發(fā)現(xiàn)肝硬化病人血清中的PDGF-C前體和TGF-β1前體含量和正常人有明顯差異。 結(jié)論：成功制備出了特異性的抗人TGF-β1和PDGF-C的兔多克隆抗體,用所制備的抗體對乙肝病毒感染的肝硬化病人和正常人血清中的Western blot檢測顯示TGF-β1和PDGF-C在肝硬化病人和正常人血清中差異明顯,表明制備的抗體對乙型肝炎病毒所導(dǎo)致的肝纖維化的分子病理機(jī)制的研究有重要價(jià)值,并對建立有效的TGF-β1和PDGF-C的檢測方法提供了前提保障。其中對PDGF-C的基因克隆和原核表達(dá)及多克隆抗體制備國內(nèi)外未見他人報(bào)道；TGF-β1的基因克隆和原核表達(dá)及多克隆抗體制備國內(nèi)已有人報(bào)道,但未見與本文TGF-β1的編碼基因部分選取相同的基因克隆和原核表達(dá)及多克隆抗體制備。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R512.62;R3416

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