抗B7-H4单链抗体库的构建和筛选及抗体特异性鉴定

发布时间：2018-03-07 21:43

本文选题：B-H　切入点：胞外区　出处：《细胞与分子免疫学杂志》2016年09期 　论文类型：期刊论文

【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溲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。
[Abstract]:Objective to construct mouse anti-B7-H4 single chain antibody (scFv) library and to screen specific antibody. Methods B7-H4 extracellular domain gene was amplified from c DNA of A549 cell line and inserted into prokaryotic expression vector p ET-28 a (). B7-H4 extracellular domain recombinant protein was obtained and purified and immunized into BALB/c mice. Total RNAs were extracted from the spleen of immunized mice. The heavy chain variable region (VHN), the light chain variable region (VH) and the V 魏 V 魏 -linker (V 魏 -linker) sequence were amplified by reverse transcription and overlapping extension of PCR- SOE-PCR. The ligation product of heavy light chain variable region gene, VH/V 魏. VH / V 魏, was ligated with p UM19-T vector and transformed into E. coli DH5 伪 competent strain. The antibody library was identified and sequenced by colony PCR technique. The constructed antibody library was translated and screened in vitro using TNT Deutzia T7 Quick for PCR DNA kit. The antibody was identified by Western blot and indirect ELISA. The correct sequence of heavy chain variable region (VH), light chain variable region (V 魏) and their conjugate products (439 BP 680bp) and 1098 BP (1098 BP) were obtained. Conclusion the mouse anti-B7-H4 extracellular ribosomal display sc Fv library was successfully constructed, and the target sc Fv was screened for binding to B7-H4 extracellular domain recombinant protein.
【作者单位】：南京师范大学生命科学学院;江苏省分子医学重点实验室;江苏省医药超分子材料及应用重点实验室;
【基金】：江苏省教育厅自然科学基金(08KJD35002)
【分类号】：R392

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