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应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.
Anti-TNF-αscFv was screened by phage display technique to construct a single-chain Fv (TNF-α) scFv library and identified by using recombinant human TNF-α (rhTNF- α) were used to amplify the mouse VH and VL genes respectively. The VH and VL genes were spliced into scFv gene by overlap extension reaction and inserted into pCANTAB 5E phagemid vector with SfiⅠ / NotⅠ site to transform into E.coli TG1 , An anti-TNF-α single-chain antibody library with a capacity of 4.6 × 108 was constructed.After three rounds of enrichment and screening of the antibody library, the antigenic specificity of the positive clones was detected by ELISA and a positive clone was obtained for sequencing analysis. The sequence of anti-TNF-αscFv gene was 774bp, encoding 258 amino acids.The recombinant plasmid was transformed into E.coli HB2151 and IPTG induced the expression of soluble scFv.The molecular weight of scFv was about 28kD by SDS-PAGE and Western blot analysis.After affinity purification The latter scFv can bind to rhTNF-α and neutralize the cytotoxicity of L929 induced by rhTNF-α.In this paper, high affinity anti-TNF-αscFv was screened by using phage antibody library, which lays the foundation for the development of new antibody for clinical immunotherapy basis.