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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TNTT7 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。
Objective To construct a murine anti-B7-H4 extracellular ribosomal scFv library and screened for high specific antibodies. Methods The extracellular domain of B7-H4 was amplified from the c DNA of A549 cells and inserted into the prokaryotic expression vector p ET-28 a (+). The recombinant protein of B7-H4 was purified and immunized BALB / c mice. Total RNA was extracted from the spleens of immunized mice and the heavy chain variable region (VH), light chain variable region (Vκ) and VH linker were amplified by reverse transcription PCR and overlap extension PCR (SOE-PCR) (VH-linker) sequence and a Vκ-linker sequence to obtain the VH / V κ of the heavy chain variable region gene. VH / Vκ was ligated with the pUC19-T vector and transformed into E. coli DH5α competent cells, which was identified and sequenced by colony PCR. The constructed antibody library was translated and screened in vitro using the TNT T7 Quick for PCR DNA kit, and the obtained antibodies were specifically identified by Western blot and indirect ELISA. Results The sequences of VH, Vκ and VH / Vκ of the correct sequences were obtained by sequencing and analysis by BioCo. The sizes were 439 bp, 680 bp and 1098 bp, respectively . After specific experimental analysis, the antibodies screened from the obtained antibody library have high specific binding ability with B7-H4. Conclusion The murine anti-B7-H4 extracellular ribosome scFv library was successfully constructed and the target sc Fv was screened for its high specificity to the recombinant extracellular domain of B7-H4.