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 the ribosome display library of anti-BT-H4 extracellular domain, and select the antibody with high specificity. Methods The cDNA of BT-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR （RT-PCR）. To express ectodomains of B7-H4, the sequence of BT-H4 gene, which encodes the BT-H4 extracellular domains, was inserted into plasmid pET-28a （ ＋ ）. 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, VK and VH/VK were amplified separately by RT-PCR and splicing by overlap extension PCR （SOE-PCR）. The gene of VH/VK was ligated into pUM19-T vector and the ligated sample was transformed into competent E. coil DH5α. 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-scFv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-BT-H4-scFv. Results The right sequences of VH, VK and VH/VK were acquired, which were 439,680 and 1098 bp in length, respectively. The analysis of specificity demonstrated that the anti-BT-H4-scFv 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-BT-H4-scFv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第9期1260-1266,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
江苏省教育厅自然科学基金(08KJD35002)
关键词
B7-H4
胞外区
单链抗体
核糖体展示
重组蛋白
B7-H4
extracellular domain
single chain antibody
ribosome display
recombinant protein
