抗人β淀粉樣肽特異性單鏈抗體的噬菌體抗體庫(kù)的構(gòu)建與初步鑒定.doc

《抗人β淀粉樣肽特異性單鏈抗體的噬菌體抗體庫(kù)的構(gòu)建與初步鑒定.doc》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫(kù)。

1、抗人β淀粉樣肽特異性單鏈抗體的噬菌體抗體庫(kù)的構(gòu)建與初步鑒定作者：段朝暉，毛越蘋，羅曉紅，李民友，勞偉思，王英，朱振宇【摘要】目的：運(yùn)用噬菌體展示技術(shù)構(gòu)建抗人β淀粉樣肽（Aβ）特異性單鏈抗體的抗體庫(kù)并進(jìn)行初步鑒定.方法：從Aβ免疫的BALB/c小鼠脾淋巴細(xì)胞中提取總RNA，分離純化mRNA，經(jīng)逆轉(zhuǎn)錄合成其總cDNA.以PCR方法分別擴(kuò)增出抗體的重鏈(VH)和輕鏈(VL)可變區(qū)基因片段，再經(jīng)重組PCR將兩基因片段由編碼十五肽(Gly4Ser)3的接頭連在一起，克隆到噬菌粒載體pCANTAB5E中，轉(zhuǎn)化至大腸桿菌TG1，經(jīng)輔助噬菌體M13KO7超感

2、染后回收全部重組噬菌體，構(gòu)建噬菌體抗體庫(kù)，SDSPAGE鑒定純度.結(jié)果：經(jīng)瓊脂糖凝膠電泳分析，RTPCR方法擴(kuò)增的抗體VH和VL基因片段大小約350bp和325bp，與預(yù)期VH，VL基因片段理論值相符.經(jīng)重組PCR得到大小約750bp的ScFv基因片段.ScFv基因片段經(jīng)雙酶切后成功定向克隆到噬菌粒載體，回收后構(gòu)建成庫(kù)容量為1.1×108的噬菌體抗體庫(kù)，經(jīng)SDSPAGE鑒定，得到Mr約為30000ku，純度較好的ScFv抗體.結(jié)論：成功構(gòu)建了庫(kù)容量大的特異性噬菌體抗體庫(kù)，為抗人Aβ特異性抗體的篩選、生物活性鑒定、臨床實(shí)驗(yàn)診斷方法建立和治療應(yīng)

3、用打下基礎(chǔ).【關(guān)鍵詞】噬菌體展示技術(shù)；單鏈抗體；阿爾茨海默??；β淀粉樣肽　　【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