spag6相互作用蛋白的酵母双杂交筛选

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1、ScreeningofProteinsInteractingwiththeSPAG6byYeastTwoHybridSystemMasterCandidate：GuanglunPingPublicHealthandPreventiveMajor：MedicineSupervisor：Prof.ZhangZBWuhanUniversityofScienceandTechnologyWuhan,Hubei430081,P.R.ChinaMay21th,2015摘要目的：构建重组SPAG6-PGBKT7质粒，用Yeast-2-

2、Hybriy实验来筛选出与SPAG6有相互作用的蛋白。方法：1.在“PubMed”数据库，寻找小鼠SPAG6全长碱基序列，分析SPAG6的活性区域（CDS区）；2.以小鼠睾丸的cDNA为模板，通过PCR技术扩增SPAG6序列；3.TA克隆；4.构建SPAG6/PGBKT7重组质粒；5.将SPAG6/PGBKT7质粒转入AH109细胞以SPAG6/PGBKT7为饵，使其与小鼠cDNA文库，进行酵母双杂交；6.提取酵母质粒；7.酵母质粒转化；8.测序后分析。结果：构建重组的SPAG6/PGBKT7质粒通过鉴定，结果正确；酵母

3、双杂交实验，初步筛选出数个与SPAG6可能发生相互作用的蛋白。结论：有许多蛋白与SPAG6有着相互作用。为进一步研究与SPAG6发生相互作用的蛋白提供依据。关键词：SPAG6；酵母双杂交；相互作用蛋白筛选IIAbstractObjective:ToexploretheproteinswhichcaninteractwithSPAG6,constructtherecombinantplasmidofSPAG6/pGBKT7andpicktheproteinsbyyeasttwohybrid.Methods:1.Lookin

4、gfortheCDSofSPAG6ofmiceinPubMed;2.UsingthetestiscDNAofmiceasamodel,amplifySPAG6sequencebyPCR;3.TAcloning;4.ConstructtherecombinantplasmidofSPAG6/PGBKT7;5.TransformSPAG6/pGBKT7intoAH109competentcell,makingyeasttwohybridwithcDNAlibraryofmice;6.Extractingplasmidsofy

5、easts;7.TransformyeastplasmidsintoDH5a;8.Sequenceanalysisoftheseplasmids.Results:TheresultsofrestrictionenzymedigestionandsequencingindicatetherecombinantplasmidofSPAG6/pGBKT7constructsuccessfully.Inyeasttwohybrid,initialscreensomeproteinswhichmayhaveinteractionw

6、ithSPAG6.Conclusion:TherearemanyproteinsmayinteractingwithSPAG6.ThisprovidesabasisforhowtoresearchtheproteinsinteractwithSPAG6.Keywords:SPAG6;yeast-2-hybrid;interactionproteinsIII目录摘要................................................................................

7、......................................................IIAbstract...............................................................................................................................III目录...................................................................

8、..............................................................IV前言.............................................................................................