噬菌体展示技术鉴定hSAMP32与hPH20抗原表位

发布时间：2018-01-13 06:37

  本文关键词：噬菌体展示技术鉴定hSAMP32与hPH20抗原表位　出处：《郑州大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： 噬菌体展示 抗原表位 抗精子抗体

【摘要】：背景与目的 抗原表位,又称抗原决定簇,是抗原分子抗原性的基础。正确而详细地绘制抗原表位图谱对疾病的诊断、设计无毒副作用的多表位疫苗以及免疫治疗试剂等具有积极的意义。噬菌体展示技术作为近年来新兴起的研究蛋白质抗原表位的有利工具,已经成功地鉴定出了多种病原微生物的抗原表位。这就为揭示一些病原体未知的抗原表位,从而进一步达到预防和控制疾病的发生提供了条件。 抗精子抗体(anti-sperm antibodies,ASAB)是男性体内的一种自身抗体,对于女性属于同种异体抗体,9%～36%的不育夫妇体内含有ASAB(男性占8%-21%,女性占6%-23%)。已证明ASAB是免疫不育的病因,同时ASAB抗体又可以用于人类避孕疫苗的研究,理想的CV精子抗原特异性表达于精子表面,涉及精子卵透明带结合,并且和人类免疫不育有关。比较显著的有受精抗原(fertilization antigen,FA-1)、透明质酸酶PH20、精子蛋白10(sperm protein,SP-10)、人精子顶体膜相关蛋白32(Human Sperm Acrosomal Membrane-AssociatedProtein32,hSAMP32)等。PH20和SAMP32都是GPI锚定的精子特异性的膜蛋白。虽然对hPH20和hSAMP32在免疫性不孕中的作用也有报道,但迄今为止,对于hPH20和hSAMP32的抗原表位还没有报道。为深入了解hSAMP32和hPH20的抗原结构,便于设计科学合理的疫苗和诊断,我们对hPH20和hSAMP32进行了抗原表位的鉴定。 材料与方法 1本研究利用分子生物学方法将hPH20和hSAMP32基因从本课题组保存的菌株中提取质粒DNA,BamHⅠ/XhoⅠ双酶切鉴定并DNA测序,将质粒DNA纯化作为模板保存。利用生物学软件Primer premier 5.0软件分别设计hSAMP32,hPH20基因的分段引物,分为Sa,Sb,Sc;Pa,Pb,Pc,Pd。所有引物上游酶切位点是EcoRI,下游是HindⅢ所用载体为T7select10-3b,各个片段经PCR扩增后与T载体连接,转化,测序。 2各个片段酶切纯化后与T7噬菌体载体相连,经体外包装后展示在T7噬菌体表面,构建了hPH20和hSAMP32基因特异性肽库。 3利用ASAB阳性血清进行了5轮筛选,淘选出5段基因片断,结合TMHMM在线分析结果。 4在Pa,Sa抗原表位所在区,经蛋白质抗原表位生物学软件分析设计合成表位肽。 5 ELISA鉴定表位肽的生物反应性,SPSS统计分析。 结果 1 Sa,Sb,Sc;Pa,Pb,Pc,Pd经测序为目的基因,分别为343bp,343bp,352bp,400bp,403bp,397bp,367bp。 2 hPH20和hSAMP32基因特异性肽库构建成功,库容为2×10~5pfu/mL。3 Pa,Sa为抗原表位所在区。 4表位肽 SAMP32: 6 AVQDAGL A H E G E G E E E T E; 37 E T E D V S N R N V V K E V E FG M; 76 E S K C V V R V E E C R G P T DC; 95 G K P I S E S L E S V R L A C I H T S; PH20: 82 KISLQDHLDKAKDITFY。 5 ELISA鉴定结果表明6号肽,37号肽,76号肽,82号肽4条表位肽的生物反应性良好。 结论 本研究初步鉴定了hPH20和hSAMP32的表位所在区,为避孕疫苗和多肽药物的开发提供了一定的理论基础。
[Abstract]:Background and purpose Antigenic epitopes, also called antigenic determinants, are the basis of antigenicity of antigens. The design of multiepitope vaccine and immunotherapeutic reagent is of great significance. Phage display is a new tool for the study of protein epitopes in recent years. The antigenic epitopes of various pathogenic microorganisms have been successfully identified, which provides conditions for the further prevention and control of diseases by revealing some unknown antigenic epitopes of pathogens. Anti-sperm antibody (anti-sperm antibodiesl ASAB) is an autoantibody in male, and belongs to allogeneic antibody to female. Nine hundred and thirty-six percent of infertile couples had Asab in their bodies (8-21 for males and 6-23for females). It has been proved that ASAB is the cause of immune infertility. At the same time, ASAB antibody can be used in the study of human contraceptive vaccine. The ideal CV sperm antigen is specifically expressed on the surface of sperm, involving the zona-binding of spermatozoa. And related to human immune infertility. The fertilization antigens FA-1 and hyaluronidase PH20 were significant. Sperm protein 10 (SP-10). Human Sperm Acrosomal Membrane-AssociatedProtein32. PH20 and SAMP32 are spermatozoa specific membrane proteins anchored by GPI, although the role of hPH20 and hSAMP32 in immunological infertility has also been reported. So far, the epitopes of hPH20 and hSAMP32 have not been reported. In order to understand the antigenic structure of hSAMP32 and hPH20. We identified the epitopes of hPH20 and hSAMP32 in order to design a scientific and reasonable vaccine and diagnose them. Materials and methods 1 the plasmid DNA was extracted by molecular biology from the strain of hPH20 and hSAMP32. BamH 鈪，

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