本文選題：人肌紅蛋白　切入點(diǎn)：全基因合成　出處：《東北林業(yè)大學(xué)》2012年碩士論文

【摘要】：為了制備用于臨床心腦血管疾病監(jiān)測(cè)的高特異性、高敏感性的抗肌紅蛋白單克隆抗體,本研究利用兩步法合成人肌紅蛋白(myoglobin,Myo)全基因的基礎(chǔ)上,構(gòu)建原核表達(dá)載體并通過BL21(DE3)表達(dá)其融合蛋白；將獲得的融合蛋白進(jìn)行His標(biāo)簽特異性親和純化。與此同時(shí),利用血清提純的人肌紅蛋白抗原篩選出高親和力的單克隆抗體,最終利用所表達(dá)的融合蛋白篩選最具特異性并且能夠進(jìn)行夾心配對(duì)的一組單克隆抗體。本研究結(jié)果和結(jié)論如下： 1.Myo全基因合成：在NCBI查詢得到Myo氨基酸序列,并將Myo密碼子經(jīng)過優(yōu)化得到大腸桿菌慣用的密碼子,反轉(zhuǎn)錄后得到Myo全序列,并利用兩步法合成Myo全基因； 2.表達(dá)載體構(gòu)建：將Myo片段并與pMD18-T Simple載體相連,得到重組質(zhì)粒Myo-pMD18-T Simple。利用Myo-pMD18-T Simple上特異性酶切位點(diǎn),將Myo片段進(jìn)行酶切膠回收,并與原核表達(dá)載體pET-28a連接,獲得可用于原核表達(dá)的重組質(zhì)粒Myo-pET-28a; 3.重組蛋白的表達(dá)和純化：利用Myo-pET-28a轉(zhuǎn)化表達(dá)型感受態(tài)細(xì)胞BL21(DE3),獲得表達(dá)菌株,并對(duì)表達(dá)條件進(jìn)行優(yōu)化。在最佳誘導(dǎo)溫度、時(shí)間、濃度下進(jìn)行IPTG誘導(dǎo)表達(dá),最終得到Myo-His融合蛋白。利用鎳柱對(duì)Myo-His融合蛋白進(jìn)行純化,經(jīng)透析濃縮得到純化蛋白； 4.單克隆抗體制備：用血清提純的人肌紅蛋白抗原免疫小鼠,經(jīng)過細(xì)胞融合及篩選得到22株分泌特異性抗體的細(xì)胞株。再經(jīng)亞克隆篩選,最終獲得5株抗體分泌量高親和力強(qiáng)的細(xì)胞株； 5.單克隆抗體鑒定：利用本實(shí)驗(yàn)中得到的Myo純化抗原經(jīng)Western Blot檢測(cè)結(jié)果顯示,所篩選的5株抗體中,除一株抗體(編號(hào)為#16-3.3)與Myo表達(dá)純抗原反應(yīng)較弱以外,其他4株抗體(編號(hào)為#14-4.3、#17-1.2、#18.2和#19-2.3)皆能與Myo表達(dá)純抗原反應(yīng),以此篩選獲得高親和力和高特異性的單克隆抗體：經(jīng)夾心ELISA檢測(cè),#17-1.2可分別與其它3株抗體進(jìn)行夾心對(duì)配。 上述研究表明,本研究成功篩選獲得高敏感性及特異性的Myo單克隆抗體。該項(xiàng)研究為建立檢測(cè)人類心臟疾病,尤其是急性心肌梗塞的臨床檢測(cè)試劑盒的制備奠定了基礎(chǔ)。此外,上述單克隆抗體可進(jìn)行夾心配對(duì)實(shí)驗(yàn),適用于未來抗體芯片的研發(fā)制備中,為一種新的聯(lián)合快速診斷心臟疾病方法的建立提供了科學(xué)依據(jù)。
[Abstract]:In order to prepare a highly specific and sensitive monoclonal antibody against myoglobin for clinical monitoring of cardiovascular and cerebrovascular diseases, a two-step method was used to synthesize human myoglobin myoglobin (Myo) gene. Prokaryotic expression vector was constructed and the fusion protein was expressed by BL21DDE3. The fusion protein was purified by His tag. At the same time, the highly affinity monoclonal antibody was screened by using the purified human myoglobin antigen. Finally, the expressed fusion protein was used to screen a group of monoclonal antibodies which were the most specific and capable of sandwich pairing. The results and conclusions of this study are as follows:. 1.Myo gene synthesis: the amino acid sequence of Myo was obtained by NCBI query, the codon of Myo was optimized to obtain the codon of Escherichia coli, the whole sequence of Myo was obtained by reverse transcription, and the whole Myo gene was synthesized by two-step method. 2. Construction of expression vector: the recombinant plasmid Myo-pMD18-T simple was obtained by ligating the Myo fragment with the pMD18-T Simple vector. The Myo fragment was recovered by restriction endonuclease digestion from Myo-pMD18-T Simple and ligated with the prokaryotic expression vector pET-28a. The recombinant plasmid Myo-pET-28a was obtained for prokaryotic expression. 3. Expression and purification of recombinant protein: the expression strain was obtained by transforming Myo-pET-28a into BL21DE-3 cells, and the expression conditions were optimized. The expression of IPTG was induced by IPTG at the best temperature, time and concentration. Finally, Myo-His fusion protein was obtained. Myo-His fusion protein was purified by nickel column and purified by dialysis. 4. Preparation of monoclonal antibodies: mice were immunized with human myoglobin antigen purified from serum. 22 cell lines secreting specific antibodies were obtained by cell fusion and screening. Finally, 5 cell lines with high affinity for antibody secretion were obtained. 5.Monoclonal antibody identification: the purified Myo antigen obtained in this experiment was detected by Western Blot. The results showed that except one antibody (#16-3.3) reacted weakly with the pure Myo antigen, The other four antibodies (#14-4.3G #17-1.2#18.2 and #19-2.3) could react with the pure antigen expressed by Myo, so as to obtain high affinity and high specificity monoclonal antibodies. #17-1.2 could be matched with the other three antibodies by sandwich ELISA. The results of the above studies indicate that this study was successful in screening highly sensitive and specific monoclonal antibodies against Myo, which were used for the detection of human heart disease. In particular, the preparation of clinical test kit for acute myocardial infarction has laid the foundation. In addition, the above monoclonal antibody can be used for sandwich pairing test, which is suitable for the preparation of antibody chip in the future. It provides a scientific basis for the establishment of a new combined rapid diagnosis method for heart disease.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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