HgACE的生物信息学分析及其抗体的制备

发布时间：2019-06-10 00:50

【摘要】：目的:利用生物信息学分析hgACE抗原表位,制备抗hgACE的多克隆抗体,为hgACE的功能研究提供实验室基础。 方法:1.利用在线资源ExPASy( http://www.expasy.org/tools )、TMHMM(http: //www.cbs.dtu.dk./services/TMHMM)、protparam ( http://www.expasy.o rg/cgi-bin/proparam )、protscale ( http://www.expasy.org/cgi-bin/pro tscale )、sppider( http://www.sppider.cchmc.org/sppider_doc.html)、SOPMA以及DNAstar(protean)生物信息学软件对hgACE的氨基酸序列分析,预测该蛋白的亲水性、抗原性、表面可及性、可塑性、二级结构以及跨膜结构,经同源性检索后,综合考虑抗体设计的基本原则,选取具有抗原性的多肽段。2.化学合成抗原多肽与钥孔戚血蓝素(keyhole limpet hemocyanin,KLH)偶联,免疫家兔制备多克隆抗体。 3.间接ELISA方法测定抗体效价,Western blot鉴定其特异性以及免疫组织化学方法对精子表面的gACE定位。 结果:(1)成功选取了具有抗原性的多肽表位,其序列为第281-291位氨基酸(YVRRALHRHYG)。(2)制备了抗hgACE的多克隆抗体。(3)ELISA测定抗体效价1:16000;Western blot证实,hgACE多克隆抗体与重组的hgACE抗原能够特异性结合;免疫组织化学染色证实利用该抗体对人精子表面gACE定位,其结果与文献报道一致。 结论:利用生物信息学软件较好地预测出hgACE的抗原表位,并成功制备了高效价、高特异性的hgACE多克隆抗体。
[Abstract]:Aim: to analyze hgACE epitopes by bioinformatics and prepare polyclonal antibodies against hgACE, so as to provide laboratory basis for the functional study of hgACE. Method: 1. Using online resources ExPASy (http://www.expasy.org/tools), TMHMM (http: / www.cbs.dtu.dk./services/TMHMM), protparam (http://www.expasy.o rg/cgi-bin/proparam), The amino acid sequence of hgACE was analyzed by protscale (http://www.expasy.org/cgi-bin/pro tscale), sppider (http://www.sppider.cchmc.org/sppider_doc.html),SOPMA and DNAstar (protean) bioinformatics software) to predict the hydrophilicity of hgACE. Antigenicity, surface accessibility, plasticity, secondary structure and transmembrane structure. After homology retrieval, considering the basic principles of antibody design, the polypeptide segment with antigenicity was selected. 2. The polyclonal antibody was prepared by chemical synthesis of antigen polypeptide coupled with keyhole hemocyanin (keyhole limpet hemocyanin,KLH). 3. Indirect ELISA assay was used to determine the specificity of antibody titer, Western blot and gACE localization on sperm surface by immunohistochemical method. Results: (1) the polypeptide epitope with antigenicity was successfully selected, and its sequence was 281-291amino acid (YVRRALHRHYG). (2). The polyclonal antibody against hgACE was prepared. (3) the titer of the antibody was 1 鈮，

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