抗人CTGF单链抗体噬菌体展示文库的构建

摘要

目的 构建抗人结缔组织生长因子(CTGF)单链抗体(scFv)噬菌体展示文库.方法 用重组人CTGF(rhCTGF)免疫balb/c小鼠,表达CTGF抗体后取小鼠脾脏获得B细胞,提取总RNA后反转录为cDNA,聚合酶链反应(PCR)获取重链可变区(vh)和轻链可变区(vl),vh和vl再次PCR装配为vl-linker-vh即scFv基因片段,并与噬菌粒PAK100酶连接,形成含有抗结缔组织生长因子单链抗体基因的目的噬菌粒；电转化入大肠杆菌XLl-blue,加入辅助噬菌体扩增后,建立噬菌体展示库重组库.结果 经过凝胶电泳及噬菌粒基因测序分析,证实成功构建了抗结缔组织生长因子单链抗体定向免疫库.结论 成功的构建了抗结缔组织生长因子单链抗体库,为制备抗结缔组织生长因子单链抗体奠定基础.%Objective To construct single chain Fv (scFv) library of human connective tissue growth factor (CTGF) by assembling the gene from immunized mice to phagemid PAK100.Methods Mice were immunized with recombinant human CTGF (rhCTGF).Total RNA was extracted from splenocytes of the immunized mice.Complementary DNA was got by reverse transcription PCR with the extracted RNA.Then the fragment of VH (variable heavy) and VL (variable light) were obtained by PCR.And they were assembled into scFv,which was inserted into phagemids vector PAK100 through SfiIsites.The phagemids containing scFV were transferred into E.coli.XL1-Blue bacterial cells to construct the scFv library to anti-human CTGF.Results Agarose gel clectrophoresis and sequences of phagemids certified that scFv phage display library to anti-human-CTGF was established.Conclusions The establishment of scFv library of anti-human CTGF set a foundation to get anti-human CTGF scFv.