【摘要】 目的构建小鼠抗B7-H4胞外区核糖体展示单链抗体(sc Fv)库,并筛选出特异性高的抗体。方法从A549细胞的c DNA中扩增B7-H4胞外区基因,将其插入原核表达载体p ET-28 a(+)中表达,获得B7-H4胞外区重组蛋白后纯化并免疫BALB/c小鼠。从免疫小鼠脾脏中提取总RNA,并利用反转录PCR、重叠延伸PCR(SOE-PCR)等技术扩增出重链可变区(VH)、轻链可变区(Vκ)、VH接头(VH-linker)序列和Vκ接头(Vκ-linker)序列,得到重轻链可变区基因的连接产物VH/Vκ。VH/Vκ与p UM19-T载体连接并转化至E.coli DH5α感受态菌株中,利用菌落PCR技术鉴定并测序。使用TNTT7 Quick for PCR DNA试剂盒对构建出的抗体库进行体外翻译与筛选,采用Western blot法和间接ELISA对得到的抗体进行特异性鉴定。结果经生物公司测序分析,得到序列正确的重链可变区(VH)、轻链可变区(Vκ)及二者连接产物(VH/Vκ),大小分别为439 bp、680 bp及1098 bp。经特异性实验分析,从得到的抗体库中筛选出的抗体与B7-H4具有高特异性结合能力。结论成功构建出鼠抗B7-H4胞外区核糖体展示sc Fv库,并筛选出与B7-H4胞外区重组蛋白高特异性结合的目标sc Fv。更多还原

【Abstract】 Objective To construct the ribosome display library of anti-B7-H4 extracellular domain,and select the antibody with high specificity. Methods The c DNA of B7-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR( RT-PCR). To express ectodomains of B7-H4,the sequence of B7-H4 gene,which encodes the B7-H4 extracellular domains,was inserted into plasmid p ET-28 a( +). The purified recombinant protein of B7-H4 extracellular domain was used to immunize BALB/c mice. The total RNA was extracted from the spleen of BALB/c mice which had been immunized with B7-H4 recombinant protein. The genes of VH,Vκand VH/Vκwere amplified separately by RT-PCR and splicing by overlap extension PCR( SOE-PCR). The gene of VH/Vκwas ligated into p UM19-T vector and the ligated sample was transformed into competent E. coli DH5 α. The resulting plasmid was isolated and then subjected to sequencing to verify the gene sequence. TNT T7 Quick for PCR DNA kit was used to translate and screen the anti-B7-H4-sc Fv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-B7-H4-sc Fv.Results The right sequences of VH,Vκand VH/Vκwere acquired,which were 439,680 and 1098 bp in length,respectively.The analysis of specificity demonstrated that the anti-B7-H4-sc Fv screened from the ribosome display library had a high specific combining ability with B7-H4. Conclusion The experiment has successively constructed the ribosome display library of anti-B7-H4 extracellular domain, and selected the anti-B7-H4-sc Fv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain.更多还原