本文關鍵詞：噬菌體展示技術鑒定hSAMP32與hPH20抗原表位　出處：《鄭州大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： 噬菌體展示 抗原表位 抗精子抗體

【摘要】：背景與目的 抗原表位,又稱抗原決定簇,是抗原分子抗原性的基礎。正確而詳細地繪制抗原表位圖譜對疾病的診斷、設計無毒副作用的多表位疫苗以及免疫治療試劑等具有積極的意義。噬菌體展示技術作為近年來新興起的研究蛋白質抗原表位的有利工具,已經成功地鑒定出了多種病原微生物的抗原表位。這就為揭示一些病原體未知的抗原表位,從而進一步達到預防和控制疾病的發(fā)生提供了條件。 抗精子抗體(anti-sperm antibodies,ASAB)是男性體內的一種自身抗體,對于女性屬于同種異體抗體,9%～36%的不育夫婦體內含有ASAB(男性占8%-21%,女性占6%-23%)。已證明ASAB是免疫不育的病因,同時ASAB抗體又可以用于人類避孕疫苗的研究,理想的CV精子抗原特異性表達于精子表面,涉及精子卵透明帶結合,并且和人類免疫不育有關。比較顯著的有受精抗原(fertilization antigen,FA-1)、透明質酸酶PH20、精子蛋白10(sperm protein,SP-10)、人精子頂體膜相關蛋白32(Human Sperm Acrosomal Membrane-AssociatedProtein32,hSAMP32)等。PH20和SAMP32都是GPI錨定的精子特異性的膜蛋白。雖然對hPH20和hSAMP32在免疫性不孕中的作用也有報道,但迄今為止,對于hPH20和hSAMP32的抗原表位還沒有報道。為深入了解hSAMP32和hPH20的抗原結構,便于設計科學合理的疫苗和診斷,我們對hPH20和hSAMP32進行了抗原表位的鑒定。 材料與方法 1本研究利用分子生物學方法將hPH20和hSAMP32基因從本課題組保存的菌株中提取質粒DNA,BamHⅠ/XhoⅠ雙酶切鑒定并DNA測序,將質粒DNA純化作為模板保存。利用生物學軟件Primer premier 5.0軟件分別設計hSAMP32,hPH20基因的分段引物,分為Sa,Sb,Sc;Pa,Pb,Pc,Pd。所有引物上游酶切位點是EcoRI,下游是HindⅢ所用載體為T7select10-3b,各個片段經PCR擴增后與T載體連接,轉化,測序。 2各個片段酶切純化后與T7噬菌體載體相連,經體外包裝后展示在T7噬菌體表面,構建了hPH20和hSAMP32基因特異性肽庫。 3利用ASAB陽性血清進行了5輪篩選,淘選出5段基因片斷,結合TMHMM在線分析結果。 4在Pa,Sa抗原表位所在區(qū),經蛋白質抗原表位生物學軟件分析設計合成表位肽。 5 ELISA鑒定表位肽的生物反應性,SPSS統(tǒng)計分析。 結果 1 Sa,Sb,Sc;Pa,Pb,Pc,Pd經測序為目的基因,分別為343bp,343bp,352bp,400bp,403bp,397bp,367bp。 2 hPH20和hSAMP32基因特異性肽庫構建成功,庫容為2×10~5pfu/mL。3 Pa,Sa為抗原表位所在區(qū)。 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鑒定結果表明6號肽,37號肽,76號肽,82號肽4條表位肽的生物反應性良好。 結論 本研究初步鑒定了hPH20和hSAMP32的表位所在區(qū),為避孕疫苗和多肽藥物的開發(fā)提供了一定的理論基礎。
[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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