1第35卷第1期2023年VOL.35NO.12023菏泽医学专科学校学报JOURNALOFHEZEMEDICALCOLLEGEDOI:10.3969/j.issn.1008-4118.2023.01.001*应用核糖体展示技术筛选抗人p53单链抗体的效果陈荫楠，罗婉妹，罗彩林，郑晨娜，郭娜燕（泉州医学高等专科学校，福建泉州362000）摘要：目的运用核糖体展示技术筛选抗人p53单链抗体，并进行初步分析。方法诱导表达p53蛋白，用其免疫小鼠。提取免疫小鼠脾脏组织的总RNA，通过反转录PCR和重叠延伸PCR分别扩增抗体重链可变区基因VH-Linker和轻链可变区基因VL-Linker，并合成VH-linker-VL型单链抗体基因。TNTT7QuickforPCRDNA试剂盒对单链抗体基因进行体外转录与翻译，以p53蛋白为靶标，经过5轮筛选富集ScFv基因。将获取单链抗体片段进行原核表达后，采用ELISA法和细胞免疫学法检测。结果成功构建抗p53单链抗体库，并筛选出p53蛋白具有较强结合活性的单链抗体株p53-scFv1、p53-scFv2和p53-scFv3，可用以检测细胞中的p53蛋白。结论利用核糖体展示技术获得亲和力较好的抗人p53单链抗体，可为p53抗体的制备提供新思路。关键词：p53蛋白；单链抗体；核糖体中图分类号：Q753文献标识码：A文章编号：1008-4118（2023）01-0001-06Screeningofanti-humanp53singlechainantibodybyribosomedisplaytechnologyCHENYinnan,LUOWanmei,LUOCailin,ZHENGChenna,GUONayan（QuanzhouMedicalCollege,Quanzhou362000,Fujian）Abstract:ObjectiveToscreenandanalyzeanti-humanp53singlechainantibodybyribosomedisplaytechnology.MethodsImmunemicewereinducedexpressionofp53protein.TotalRNAwasextractedfromspleentissuesofimmunizedmice.TheVH-LinkerandVL-LinkergeneswereamplifiedbyreversetranscriptionPCRandoverlappingextensionPCRrespectively,andVH-Linker-VLtypeSCFVgenesweresynthesized.TNTT7QuickforPCRDNAkitwasusedintranscriptionandtranslationofsinglechainantibodygene,withp53proteinasthetargetSCFVgenewasenrichedafterfiveroundsofscreening.Afterprokaryoticexpression,theSCFVfragmentsweredetectedbyELISAandcellularimmunoassay.ResultsAnti-p53singlechainantibodylibrarywassuccessfullyconstructed.Thesinglechainantibodystrainsp53-scFv1,p53-scFv2andp53-scFv3withstrongbindingactivitywerescreenedouttodetectp53proteinincells.ConclusionTheribosomedisplaytechnologywasusedtoobtainanti-humanp53SCFVw...
