胰腺多肽纳米抗体的制备及结合活性分析  

Preparation of Pancreatic Polypeptide Nanobody and the Analysis of Binding Activity

作　　者：任泓睿 贾琼 王家庆 田婧婧 李荣杰 郝花花 李建丽 渠志灿 范瑞文[1] REN Hong-Rui;JIA Qiong;WANG Jia-Qin;TIAN Jing-Jing;LI Rong-Jie;HAO Hua-Hua;LI Jian-Li;QU Zhi-Can;FAN Rui-Wen(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China;Nanolattix Biotechnology Co,Ltd,Taiyuan 030006,Shanxi,China)

机构地区：[1]山西农业大学动物医学学院羊驼生物工程实验室,山西晋中030801 [2]山西纳安生物科技股份有限公司,太原030006

出　　处：《中国生物化学与分子生物学报》2024年第9期1273-1281,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：山西省重点研发计划(大健康与生物医药领域)(No.202202130501005)资助。

摘　　要：胰腺多肽(pancreatic polypeptide,PP)是一种含36个氨基酸的胰腺激素,在评估胰腺功能和辅助诊断相关疾病中发挥重要作用。以PP为靶点,筛选特异性的PP纳米抗体,评估其与PP抗原是否具有结合活性。利用原核表达系统制备了高纯度的PP抗原,经免疫羊驼后构建了高库容和高丰度的胰腺多肽免疫的纳米抗体噬菌体文库,并通过噬菌体展示技术进行文库筛选,获得8株胰腺多肽纳米抗体。选取1株纳米抗体(PP-VHH)构建原核表达体系,经IPTG诱导表达、纯化与鉴定,通过间接ELISA和双抗夹心ELISA分别检测和评价抗原抗体的结合活性和血清中PP含量,结果显示,PP-VHH与PP具有结合活性,且PP-VHH可用于检测鸡和人血清中的PP。建立的双抗夹心ELISA法拟合曲线R^(2)为0.9868,可检测不同个体鸡血清中PP浓度为48~55 pg/mL。而人血清中PP浓度范围波动较大。综上所述,本研究筛选到的1株纳米抗体PP-VHH,可用于检测鸡和人血清中的PP含量,为评估胰腺功能异常或诊断相关疾病提供基础。Pancreatic polypeptide(PP),a pancreatic hormone containing 36 amino acids,plays important roles in the diagnosis and evaluation of pancreatic function,injury and diseases.In this study,a phage nanobody library against PP was constructed to screen specific PP nanobodies,which would be used to evaluate whether they have binding activity with PP antigen.After PP antigen with high purity was prepared by prokaryotic expression system,it was used to immunize alpaca to construct the nanobody library against PP with high storage capacity and high abundance,from which 8 strains of PP nanobodies were obtained by phage display.One of nanobody strain(PP-VHH)was selected to be expressed in a prokaryotic expression system,which was induced overnight by IPTG.After purification and identification,the antigen-antibody binding activity and PP level in serum were detected by indirect ELISA and Sandwich ELISA methods,respectively.The results showed that PP-VHH had binding activity with PP,which could be used to detect PP in chicken and human serum.The Sandwich ELISA methods with R^(2)of the fitting curve 0.9868 could be used to detect PP concentrations of 48-55 pg/mL in the serum of chickens,while the concentrations of PP in human serum varied significantly.In summary,PP-VHH screened from nanobody library against PP could detect PP in serum,which would supply the basis for evaluation of abnormal pancreatic function and diagnosis of relative disease.

关 键 词：胰腺多肽 纳米抗体 原核表达 结合活性 

分 类 号：Q812[生物学—生物工程]