抗肝癌单链抗体基因库的制备

Construction of single-chain Fv library against hepatocellular carcinoma

目的构建抗肝癌噬菌体单链抗体(Single-chainvariblefragments,ScFv)库并加以鉴定。方法从经肝癌细胞免疫小鼠脾淋巴细胞中提取mRNA,RT-PCR扩增轻、重链可变区基因片段,连接成ScFv基因并扩增,克隆到pCANTAB5E,导入大肠杆菌TG1培养并计数。PCR检测ScFv基因插入情况并对ScFv进行基因序列分析和ELASA检测。结果抗体库库容为2.14×106;90%的噬菌体基因中有ScFv基因的插入;核酸序列分析显示抗体库基因结构正确;ELASA检测证实ScFv的表达。结论所建抗肝癌噬菌体ScFv库库容大,质量良好,对肝癌的基础研究和临床有潜在的应用价值。

Objective To constructa phagedisplaysingle-chainFv(ScFv)libraryagainsthepatocellularcarcinoma(HCC).Methods The mRNAextractedfromthespleencellsof immunizedBALB/Cmicewas purifiedand reversetranscriptase-polymerasechainreaction(RT-PCR)was employedfortheseperateamplificationof thegenefragmentsencodingtheheavy-andlight-chainvariableregionsrespectively,whichweresubsequentlyassembledintoScFvgene.Amplificationof the resultantScFv gene was performedbeforeit was introducedintoE.coli TG1via a phagemidvectorpCANTAB5E.A proportionof the transformedcellswas loadedonto SOBAGplatesand incubatedand the numberof bacterialcolonies counted.PCR,nucleotidessequenceanalysisandenzyme-linkedimmunosorbentassay(ELISA)wereperformedto evaluate theconstructedScFvlibrary.Result ThephagedisplayScFvlibraryhada capacityof approximately2.14×10 6 ,with90%of the phagemidscontainingScFv gene insertionas demonstratedby PCR.The nucleotidessequenceof ScFv gene was approved,andELISAshowedthattheScFvlibrarycouldspecificallyconjugatewithHCCantigen.Conclusion Thephage displayScFvlibraryagainstHCCis successfullyconstructed,whichmayfacilitatetheacquisitionof specialantibodiesand maybe instrumentalintheresearchandclinicalmanagementof HCC.