摘要
为制备鼠抗鸡膜联蛋白A2(ANXA2)多克隆抗体,检测ANXA2蛋白在鸡不同组织中的表达情况,先构建鸡ANXA2基因的重组原核表达载体pET-32a-ANXA2,将其转化入大肠埃希菌中进行诱导表达;采用切胶纯化和切胶免疫小鼠的方法制备鸡ANXA2多克隆抗体,然后利用该抗体检测ANXA2蛋白在4周龄SPF鸡不同组织中的表达情况。结果表明:鸡ANXA2重组蛋白His-ANXA2在IPTG终浓度为0.1 mmol·L-1、诱导时间为5 h的条件下表达量高。通过切胶纯化和切胶免疫方法获得了具有较高效价和特异性的鸡ANXA2蛋白鼠多克隆抗体。组织表达检测结果显示,ANXA2蛋白在鸡法氏囊、胸腺、十二指肠、肾脏、脑、腿肌和心脏中有表达,而在肝脏、脾脏、肺脏、腺胃和胰腺中未检测到表达。这一研究获得的鼠抗鸡ANXA2蛋白多克隆抗体为后续研究鸡相关病毒的复制和致病机制奠定了基础。
This study aimed to prepare the mouse polyclonal antibody against chicken annexin A2(ANXA2)and detect the tissue expression of ANXA2 protein.The recombinant plasmid pET-32 a-ANXA2 was constructed and then transfected into E.coli to express recombinant protein His-ANXA2.The gel-cutting purification and gel-cutting immunity methods were performed to prepare the mouse polyclonal antibody against chicken ANXA2,which were then used to detect the expression of ANXA2 protein in different tissues of 4-week-old SPF chickens.The results showed that the recombinant protein His-ANXA2 was relatively high expressed under the induction condition of 0.1 mmol·L;IPTG and 5 h.The obtained mouse polyclonal antibody against chicken ANXA2 had high titer and high specificity.In addition,the tissue expression of ANXA2 revealed that ANXA2 protein was detected in the bursa of fabricius,thymus,duodenum,kidney,brain,crureus and heart,but not in liver,spleen,lung,glandular stomach and pancreas.This study will provide a foundation for further studying the replication and pathogenicity of some related chicken viruses.
作者
高洪波
袁超
周磊
韩一帆
段志强
GAO Hongbo;YUAN Chao;ZHOU Lei;HAN Yifan;DUAN Zhiqiang(Key Laboratory of Animal Genetics,Breeding and Reproduction in The Plateau Mountainous Region,Ministry of Education/Key Laboratory of Animal Genetics,Breeding and Reproduction in The Plateau Mountainous Region of Guizhou Province,Guizhou University,Guiyang 550025,China;College of Animal Science,Guizhou University,Guiyang 550025,China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2021年第4期23-28,共6页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
国家自然科学基金资助项目(31760732、31960698)
贵州省基础研究计划(科学技术基金)项目(黔科合基础[2020]1Y134)。
关键词
鸡
膜联蛋白A2
多克隆抗体
原核表达
chicken
ANXA2
polyclonal antibody
prokaryotic expression
