心脏型脂肪酸结合蛋白（H-FABP）的原核表达、纯化及其单克

发布时间：2018-11-24 09:16

【摘要】：通过RT-PCR扩增人心脏型脂肪酸结合蛋白H-FABP的基因片断，将目的基因与原核表达载体pET28a连接，构建原核表达重组质粒pET28a-H-FABP。将重组质粒转化到大肠杆菌BL21中，通过IPTG诱导表达人心脏型脂肪酸结合蛋白。利用pET28a构建的H-FABP原核表达质粒在BL21实现了高效表达，经SDS-PAGE电泳分析显示，表达的重组蛋白分子质量约为15kD。采用离子交换层析柱纯化H-FABP重组蛋白，通过Q-HP柱（pH11.5）纯化后得到高纯度H-FABP重组蛋白，含量约为0.48mg/mL。Western blotting鉴定纯化后重组蛋白特异性结果表明，所得重组蛋白与商品化的H-FABP单抗呈特异性反应。利用B细胞表位在线预测软件，综合分析蛋白结构β-转角，蛋白抗原表面可及性，蛋白柔韧性，蛋白抗原性，疏水性及线性抗原表位，我们选择了具有较强抗原特异性的H-FABP区段作为免疫原与重组H-FABP一起免疫动物。通过改良免疫方法将H-FABP全长抗原及表位肽免疫新西兰大白兔获得兔抗人H-FABP抗血清，应用蛋白A柱将抗血清中IgG组分纯化分离出来，应用亲和层析技术将抗体中针对重组蛋白标签的抗体去除，得到高特异针对H-FABP的兔抗人多克隆抗体。通过ELISA，Western blotting等方法证实了该抗体的高效价和高特异性。使用重组H-FABP全长抗原及表位肽免疫Balb/c小鼠，应用改良的杂交瘤技术将免疫小鼠的脾细胞与Balb/c小鼠骨髓瘤细胞Sp2/0在聚乙二醇作用下融合，，HAT选择性培养，间接酶联免疫吸附方法对融合细胞培养上清进行筛选，选择抗H-FABP单克隆抗体阳性的细胞株采用有限稀释法连续进行亚克隆，建立稳定分泌抗H-FABP单克隆抗体的杂交瘤细胞株。采用小鼠腹腔内诱生法扩大培养阳性单克隆细胞株后，腹腔注射石蜡处理过的小鼠，制备腹水。腹水经HiTrap IgG PurificationHP亲和层析柱纯化，所得的单克隆抗体进行效价测定，亲和力测定。经筛选得到6株稳定分泌抗H-FABP单克隆抗体的杂交瘤细胞株，经亚类鉴定均为IgG型，6株单抗效价均在1：256000以上，亲和力最高可达9.02×10~(-9)。Western blotting鉴定表明6株单克隆抗体均具有较高的特异性。成功制备的这6株抗H-FABP特异性的单克隆抗体，可应用于建立一种新型H-FABP免疫检测方法。利用实验室已制备的抗H-FABP抗体作为捕获抗体和检测抗体，以H-FABP重组蛋白作为检测抗原,进行抗体配对试验，经过优化ELISA反应条件，分别检测26例临床AMI血清及34例正常血清标本中的H-FABP，并进行结果比较分析，结果成功得到了能检测天然H-FABP的配对抗体3-H5—1-F10，并以此成功建立了检测H-FABP的双抗体夹心ELISA方法。因此，所获得的高特异性抗体3-H5—1-F10将会为今后H-FABP的检测以及新型免疫检测试剂盒的研发奠定重要的基础。
[Abstract]:The gene fragment of human heart fatty acid binding protein (H-FABP) was amplified by RT-PCR. The target gene was ligated with prokaryotic expression vector pET28a to construct the prokaryotic expression plasmid pET28a-H-FABP.. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by IPTG to express human cardiac fatty acid binding protein. The prokaryotic expression plasmid of H-FABP constructed by pET28a was highly expressed in BL21. The molecular weight of the expressed recombinant protein was about 15kD by SDS-PAGE electrophoresis. The recombinant H-FABP protein was purified by ion exchange chromatography and purified by Q-HP column (pH11.5). The high purity H-FABP recombinant protein was obtained. The specific content of the recombinant protein was identified by 0.48mg/mL.Western blotting. The recombinant protein reacted specifically with the commercial H-FABP monoclonal antibody. B cell epitope on-line prediction software was used to analyze the 尾 -rotation angle of protein structure, surface accessibility of protein antigen, protein flexibility, protein antigenicity, hydrophobicity and linear antigen epitope. We selected H-FABP region with strong antigen specificity as immunogen to immunize animals with recombinant H-FABP. Rabbit anti-human H-FABP antiserum was obtained by immunizing New Zealand white rabbits with H-FABP full-length antigen and epitope peptide by modified immune method. The IgG component in the antiserum was purified by protein A column. Rabbit anti-human polyclonal antibodies with high specificity to H-FABP were obtained by using affinity chromatography to remove the antibodies against recombinant protein labels. The high titer and specificity of the antibody were confirmed by ELISA,Western blotting and other methods. Balb/c mice were immunized with recombinant H-FABP full-length antigen and epitope peptide. The spleen cells of the immunized mice were fused with Balb/c mouse myeloma cells Sp2/0 under the action of polyethylene glycol and HAT was selectively cultured by modified hybridoma technique. Indirect enzyme-linked immunosorbent assay (Elisa) was used to screen the supernatant of the fusion cell culture. Subclones of the cell lines that were positive for H-FABP monoclonal antibody were subcloned by limited dilution method. To establish a hybridoma cell line secreting monoclonal antibody against H-FABP stably. Ascites were prepared by intraperitoneal injection of paraffin-treated mice. Ascites were purified by HiTrap IgG PurificationHP affinity chromatography. Six hybridoma cell lines stably secreting monoclonal antibodies against H-FABP were obtained. All of them were identified as IgG type by subclass identification, and the titers of 6 McAbs were above 1: 256000. The highest affinity of 9. 02 脳 10 ~ (-9). Western blotting showed that all of the 6 McAbs had high specificity. The six monoclonal antibodies against H-FABP can be used to establish a new H-FABP immunoassay. The anti H-FABP antibody prepared in the laboratory was used as the capture antibody and the detection antibody, and the recombinant H-FABP protein was used as the detection antigen. The antibody pairing test was carried out, and the conditions of ELISA reaction were optimized. H-FABPs were detected in 26 clinical AMI sera and 34 normal serum samples, and the results were compared and analyzed. The results showed that the paired antibodies 3-H5-1-F10, which could detect natural H-FABP, were successfully obtained. A double antibody sandwich ELISA method for the detection of H-FABP was successfully established. Therefore, the obtained high specific antibody 3-H5-1-F10 will lay an important foundation for the detection of H-FABP and the development of new immunoassay kit in the future.
【学位授予单位】：重庆师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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