犬NT-proCNP单链抗体库的构建及1株单链抗体的筛选与免疫活性检测  

Establishment of single-chain antibody library targeting canine NT-proCNP,and screening and immune activity detection of a selected single-chain antibody

作　　者：江少佳 南沙 王慧康 毛玲 尹瑞玲 雷蔷会 王浩龙 李好 萧锦瑜 丁明星[1] 丁一 JIANG Shaojia;NAN Sha;WANG Huikang;MAO Ling;YIN Ruiling;LEI Qianghui;WANG Haolong;LI Hao;XIAO Jinyu;DING Mingxing;DING Yi(College of Animal Sciences and Technology/College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430000,China)

机构地区：[1]华中农业大学动物科学技术学院/动物医学院,湖北武汉430000

出　　处：《中国兽医学报》2025年第3期535-541,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32072938)。

摘　　要：首先采用原核表达制备氨基末端C型钠尿肽原(NT-proCNP)并对新西兰大白兔进行免疫。当兔血清效价达到预期后,提取其脾细胞总RNA,并反转录为cDNA。以cDNA为模板,利用简并引物分别对兔源抗体轻链可变区(VL)以及重链可变区(VH)基因进行扩增。通过重叠延伸PCR法将VL和VH拼接成完整的噬菌体单链抗体(scFv)片段,再将scFv与嗜菌粒pComb3XSS连接并电转化至感受态E.coli TG1中以构建抗NT-proCNP兔源单链抗体免疫文库。对该抗体库进行4轮富集淘筛,并从中筛选出特异性单链抗体。最后通过原核表达系统对单链抗体进行可溶性表达并检测其免疫反应活性。结果表明,本研究基于噬菌体展示技术成功筛选到1株具有良好抗原结合活性且符合scFv基因特征的单链抗体scFv-1-CNP,为进一步探究单链抗体在NT-proCNP检测中的应用提供了研究思路。The amino-terminal pro-C-type natriuretic peptide(NT-proCNP)is a diagnostic inflammatory marker clinically used for diagnosing bacterial infections.This study aims to establish a phage display library of single-chain variable fragment(scFv)antibodies against canine NT-proCNP and to screen for scFvs with high binding affinity to NT-proCNP.Initially,NT-proCNP was prepared using prokaryotic expression system and was used to immunize New Zealand White rabbits.Upon achieving the desired serum titer,total RNA was extracted from the splenocytes of rabbits and reverse transcribed into cDNA.Using this cDNA as a template,degenerate primers were employed to amplify the genes of the rabbit antibody light chain variable region(VL)and heavy chain variable region(VH).The VL and VH regions were spliced together to form a complete scFv fragment via overlap extension PCR.The scFv was then ligated into the phagemid pComb3XSS and electroporated into competent E.coli TG1 cells to construct a rabbit-derived antiNT-proCNP scFv immunological library.This library underwent four rounds of enrichment and screening to isolate specific single-chain antibodies.The selected antibody was subsequently expressed in a soluble form within a prokaryotic system,and its immunological activity was evaluated.Using phage display technology,this study successfully identified a single-chain antibody scFv-1-CNP with strong antigen-binding activity and genetic sequence characteristics of scFvs,providing a research direction for further exploration of scFv applications in the detection of NTproCNP.

关 键 词：氨基末端C型钠尿肽原 单链抗体 噬菌体展示 

分 类 号：S852.4[农业科学—基础兽医学]