鸡DEC-205结合肽的筛选及体外鉴定  

Screening and identification of chicken DEC-205 binding peptidein in vitro

作　　者：马孙婷 谭飞 王晓丽[1] 马莉莉 徐彬 吕立新[1] 冯志新[1] 陈佩佩 欧阳伟[1,4] MA Sunting;TAN Fei;WANG Xiaoli;MA Lili;XU Bin;LYU Lixin;FENG Zhixin;CHEN Peipei;OUYANG Wei(Ministry of Agriculture Key Laboratory of Veterinary Biological Engineering and Technology,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Agricultural Com prehensive Service Center of Lyusigang Town of Qidong City,Qidong,Jiangsu 226200,China;Animal Husbandry and Veterinary Station,Sucheng District of Suqian City,Suqian,Jiangsu 223800,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院,兽医研究所农业部兽用生物制品工程技术重点实验室,江苏南京210014 [2]启东市吕四港镇农业综合服务中心,江苏启东226200 [3]宿迁市宿城区畜牧兽医站,江苏宿迁223800 [4]南京农业大学动物医学学院,江苏南京210014

出　　处：《中国兽医学报》2023年第6期1270-1276,共7页Chinese Journal of Veterinary Science

基　　金：江苏省农业科技自主创新资金资助项目(CX(20)3095);国家自然科学基金资助项目(31772723,32072872)。

摘　　要：为筛选鸡DEC-205的优势结合肽,以小鼠CD8α信号肽、鸡DEC-205具有结合作用的3个C型凝集素样结构域(CTLD-4、CTLD-5和CTLD-6)、人IgG1 Fc和His标签作为目的基因,设计构建重组表达质粒pcDNA3.1-DEC-205-Fc-his和pcDNA3.1-Fc-his(用于对照);转染293t细胞,Western blot鉴定蛋白表达;镍亲和层析法纯化蛋白,用于噬菌体线性12肽库筛选结合肽,protein G/A交替进行了3轮液相淘选,对第3轮噬斑提取DNA测序并统计肽重复率。对重复率较高的短肽进行合成,ELISA及荧光显微镜观察鉴定肽与重组蛋白的结合效果。Western blot结果显示,转染重组质粒的293t细胞裂解液和细胞上清均可表达目的蛋白,DEC-205-Fc-his蛋白约90 kDa, Fc-his蛋白约35 kDa,与预期大小符合,纯化的蛋白作为靶标进行3轮淘选后,15条序列出现重复,其中DLPVNSVIFPFR(DL)重复率最高(达到6.41%)。ELISA及荧光检测结果显示,合成的DL短肽可结合表达DEC-205-Fc-his的细胞,与表达Fc-his的细胞亲和性较低。筛选获得的DL短肽经体外鉴定,结果显示是鸡DEC-205的潜在配体,相关结果将为研发鸡DEC-205新型靶向策略奠定基础。In order to obtain the dominant binding peptides of chicken DEC-205,a mouse CD8αsignal peptide,three C-type lectin-like domains(CTLD-4,CTLD-5,CTLD-6)with binding activity in chicken DEC-205,human IgG1 Fc and His tags were used as target genes.Recombinant expression plasmids(pcDNA3.1-DEC 205-Fc-his,pcDNA3.1-Fc-his used as the control)were constructed and transfected into 293T cells.The expressed protein was identified by Western blot and purified by nickel affinity chromatography.Phage linear 12-peptide library was used to screen for binding peptides of DEC-205-Fc-his protein.Three rounds of liquid phase panning were carried out by using protein G and protein A alternately.The DNA of plaques in the third round was extracted and sequenced.Each repetition rate of peptides was counted.The peptide with the highest repetition rate was synthesized.The ability of the peptide binding to the recombinant protein was identified by ELISA and fluorescence microscopy.Western blot results showed that both the lysate and supernatant of 293t cells transfected with recombinant plasmids expressed the protein of interest.As expected,DEC-205-Fc-his protein was about 90 kDa and Fc-his protein was about 35 kDa.After the purification,C-205-Fc-his protein was used as the target for three rounds of panning,15 sequences were duplicated,of which DLPVNSVIFPFR(DL)had the highest repeat rate(6.41%).The results of ELISA and fluorescence detection showed that the synthesized DL peptide could bind to cells expressing DEC-205-Fc-his,and had low affinity with cells expressing Fc-his.The DL peptide screened in this study was identified as one of the ligands of the three CTLD domains belonging to chicken DEC-205 in vitro.The relevant results will be the preliminary basis for the development of the new targeting strategy for chicken DEC-205.

关 键 词：鸡DEC-205 结合肽 噬菌体展示12肽库 液相淘选 

分 类 号：S852.2[农业科学—基础兽医学]