人肌红蛋白基因合成、原核表达及单克隆抗体制备

发布时间：2018-03-25 10:46

本文选题：人肌红蛋白　切入点：全基因合成　出处：《东北林业大学》2012年硕士论文

【摘要】：为了制备用于临床心脑血管疾病监测的高特异性、高敏感性的抗肌红蛋白单克隆抗体,本研究利用两步法合成人肌红蛋白(myoglobin,Myo)全基因的基础上,构建原核表达载体并通过BL21(DE3)表达其融合蛋白；将获得的融合蛋白进行His标签特异性亲和纯化。与此同时,利用血清提纯的人肌红蛋白抗原筛选出高亲和力的单克隆抗体,最终利用所表达的融合蛋白筛选最具特异性并且能够进行夹心配对的一组单克隆抗体。本研究结果和结论如下： 1.Myo全基因合成：在NCBI查询得到Myo氨基酸序列,并将Myo密码子经过优化得到大肠杆菌惯用的密码子,反转录后得到Myo全序列,并利用两步法合成Myo全基因； 2.表达载体构建：将Myo片段并与pMD18-T Simple载体相连,得到重组质粒Myo-pMD18-T Simple。利用Myo-pMD18-T Simple上特异性酶切位点,将Myo片段进行酶切胶回收,并与原核表达载体pET-28a连接,获得可用于原核表达的重组质粒Myo-pET-28a; 3.重组蛋白的表达和纯化：利用Myo-pET-28a转化表达型感受态细胞BL21(DE3),获得表达菌株,并对表达条件进行优化。在最佳诱导温度、时间、浓度下进行IPTG诱导表达,最终得到Myo-His融合蛋白。利用镍柱对Myo-His融合蛋白进行纯化,经透析浓缩得到纯化蛋白； 4.单克隆抗体制备：用血清提纯的人肌红蛋白抗原免疫小鼠,经过细胞融合及筛选得到22株分泌特异性抗体的细胞株。再经亚克隆筛选,最终获得5株抗体分泌量高亲和力强的细胞株； 5.单克隆抗体鉴定：利用本实验中得到的Myo纯化抗原经Western Blot检测结果显示,所筛选的5株抗体中,除一株抗体(编号为#16-3.3)与Myo表达纯抗原反应较弱以外,其他4株抗体(编号为#14-4.3、#17-1.2、#18.2和#19-2.3)皆能与Myo表达纯抗原反应,以此筛选获得高亲和力和高特异性的单克隆抗体：经夹心ELISA检测,#17-1.2可分别与其它3株抗体进行夹心对配。上述研究表明,本研究成功筛选获得高敏感性及特异性的Myo单克隆抗体。该项研究为建立检测人类心脏疾病,尤其是急性心肌梗塞的临床检测试剂盒的制备奠定了基础。此外,上述单克隆抗体可进行夹心配对实验,适用于未来抗体芯片的研发制备中,为一种新的联合快速诊断心脏疾病方法的建立提供了科学依据。
[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.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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