【摘要】：目的:利用生物信息學分析hgACE抗原表位,制備抗hgACE的多克隆抗體,為hgACE的功能研究提供實驗室基礎。 方法:1.利用在線資源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)生物信息學軟件對hgACE的氨基酸序列分析,預測該蛋白的親水性、抗原性、表面可及性、可塑性、二級結構以及跨膜結構,經(jīng)同源性檢索后,綜合考慮抗體設計的基本原則,選取具有抗原性的多肽段。2.化學合成抗原多肽與鑰孔戚血藍素(keyhole limpet hemocyanin,KLH)偶聯(lián),免疫家兔制備多克隆抗體。 3.間接ELISA方法測定抗體效價,Western blot鑒定其特異性以及免疫組織化學方法對精子表面的gACE定位。 結果:(1)成功選取了具有抗原性的多肽表位,其序列為第281-291位氨基酸(YVRRALHRHYG)。(2)制備了抗hgACE的多克隆抗體。(3)ELISA測定抗體效價1:16000;Western blot證實,hgACE多克隆抗體與重組的hgACE抗原能夠特異性結合;免疫組織化學染色證實利用該抗體對人精子表面gACE定位,其結果與文獻報道一致。 結論:利用生物信息學軟件較好地預測出hgACE的抗原表位,并成功制備了高效價、高特異性的hgACE多克隆抗體。
[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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