抗人β淀粉样肽特异性单链抗体的噬菌体抗体库的构建与初步鉴定.doc

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1、抗人β淀粉样肽特异性单链抗体的噬菌体抗体库的构建与初步鉴定作者：段朝晖，毛越苹，罗晓红，李民友，劳伟思，王英，朱振宇【摘要】目的：运用噬菌体展示技术构建抗人β淀粉样肽（Aβ）特异性单链抗体的抗体库并进行初步鉴定.方法：从Aβ免疫的BALB/c小鼠脾淋巴细胞中提取总RNA，分离纯化mRNA，经逆转录合成其总cDNA.以PCR方法分别扩增出抗体的重链(VH)和轻链(VL)可变区基因片段，再经重组PCR将两基因片段由编码十五肽(Gly4Ser)3的接头连在一起，克隆到噬菌粒载体pCANTAB5E中，转化至大肠杆菌TG1，经辅助噬菌体M13KO7超感

2、染后回收全部重组噬菌体，构建噬菌体抗体库，SDSPAGE鉴定纯度.结果：经琼脂糖凝胶电泳分析，RTPCR方法扩增的抗体VH和VL基因片段大小约350bp和325bp，与预期VH，VL基因片段理论值相符.经重组PCR得到大小约750bp的ScFv基因片段.ScFv基因片段经双酶切后成功定向克隆到噬菌粒载体，回收后构建成库容量为1.1×108的噬菌体抗体库，经SDSPAGE鉴定，得到Mr约为30000ku，纯度较好的ScFv抗体.结论：成功构建了库容量大的特异性噬菌体抗体库，为抗人Aβ特异性抗体的筛选、生物活性鉴定、临床实验诊断方法建立和治疗应

3、用打下基础.【关键词】噬菌体展示技术；单链抗体；阿尔茨海默病；β淀粉样肽　　【Abstract】AIM:ToconstructandidentifythespecificantihumanβamyloidpeptideScFvbyphageantibodylibrarydisplaysystem.METHODS:TotalRNAwasextractedfromspleenBcellsofBALB/cmiceimmunizedwithβamyloidpeptide,andmRNAwaspurifiedfromthetotalRNA,then

4、thefirststrandcDNAwassynthesizedbyreversetranscription.AntibodyVHandVLgenefragmentswereamplifiedbyprimaryPCRandtheamplifiedVHandVLPCRfragmentswerefurtherjoinedintoaScFvgenewitha15peptidelinkerDNA(Gly4Ser)3withfillinreactions.ThentheassembledScFvgenefragmentswereamplifiedan

5、drestrictionsiteswereaddedbythesecondPCR.ThedigestedScFvgenefragmentswerefinallyligatedwiththephagemidspCANTAB5EandthephagemidscontainingScFvgeneweretransformedintocompetentE.coliTG1cells.ThetransformedcellsweretheninfectedwithM13KO7helperphagetorescueallrecombinantphagemidsw

6、ithScFvgeneinsert,thusanimmunephageScFvlibrarywasconstructed.PurityofScFvwasanalyzedbySDSPAGE.RESULTS：AntibodyVHandVLgenefragmentsamplifiedbyprimaryPCRwereabout350bpand325bpafteranalysisbyagarosegelelectrophoresisandmatchedtheexpectancyofVHandVLgenefragments.ThepurifiedVHand

7、VLPCRfragmentswerefurtherjoinedintoaScFvgenewithfillinreactionsandreachedthedesired750bpexpectancy.The6recombinantphagemidwithScFvgeneinsertwasrescued,andanimmunephageScFvlibrarywiththecontentof1.1×108wassuccessfullyconstructed.AfteranalysisbySDSPAGE,apurifiedScFvgenewasgot

8、witharelativemolecularweightofabout30000u.CONCLUSION:AnimmunephageSc