摘要
目的原核表达人腺病毒(HAdV)7型DNA结合蛋白(DBP),制备DBP蛋白多克隆抗体。方法合成HAdV-7 DBP基因并克隆至pET30a载体,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导进行原核表达。经镍离子金属螯合层析纯化后免疫家兔制备多克隆抗体,使用间接酶联免疫吸附试验(ELISA)测定该抗体效价;使用Western blotting方法与间接免疫荧光方法(IFA)检测抗体特异性。以rDBP包被ELISA反应板使用间接ELISA对HAdV感染儿童急性期血清中抗DBP蛋白IgM和IgG抗体进行检测。结果成功构建HAdV-7 DBP原核表达载体,表达的重组DBP蛋白经镍柱亲和层析纯化后纯度>95%,间接ELISA方法测得制备的多克隆抗血清的抗体效价为1∶1024000,Western blotting方法和IFA方法显示该抗体具有较高的特异性,间接ELISA方法检测HAdV感染儿童急性期血清中抗DBP蛋白IgM和IgG抗体的阳性率分别为50.0%(19/38)和63.2%(24/38)。结论成功构建HAdV-7 DBP蛋白的原达表达载体,获得了HAdV-7型DBP重组蛋白和高效价的兔多克隆抗体。
Objective To express DNA-binding protein(DBP)of human adenovirus(HAdV)type 7 using the prokaryotic expression system,and product anti-HAdV-7 DBP rabbit polyclonal antibody.Methods The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a,and the recombinant plasmid was transformed into E.coli BL21(DE3)competent cell.The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside(IPTG)and purified with Ni-NTA affinity column.The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA,and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay(IFA).In addition,purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV.Results The HAdV-7 DBP plasmid was constructed successfully.The purified recombinant DBP was more than 95%after purification.The titer of polyclonal antibody was 1∶1024000.The polyclonal antibody showed high specificity in vitro using Western blotting and IFA.The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0%(19/38)and 63.2%(24/38),respectively,using indirect ELISA.Conclusion In summary,the HAdV-7 rDBP is expressed using prokaryotic expression system,and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.
作者
朱云
张林琳
段亚丽
谢正德
Zhu Yun;Zhang Linlin;Duan Yali;Xie Zhengde(Laboratory of Infection and Virology,Beijing Pediatric Research Institute,Beijing Key Laboratory of Pediatric Respiratory Infection Diseases,Research Unit of Critical Infection in Children,Chinese Academy of Medical Sciences,Beijing Children′s Hospital,Capital Medical University,Key Laboratory of Major Diseases in Children,Ministry of Education,National Clinical Research Center for Respiratory Diseases,National Center for Children′s Health,Beijing 100045,China;Laboratory of Infection and Virology,Beijing Children′s Hospital,Capital Medical University,National Center for Children′s Health,Beijing 100045,China)
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2022年第2期171-177,共7页
Chinese Journal of Preventive Medicine
基金
北京市自然科学基金项目(7204258)
首都卫生发展科研专项(首发2021-1G-3012)
国家自然科学基金(82072266)
中国医学科学院医学与健康科技创新工程项目(2019-I2M-5-026)。
关键词
人腺病毒7型
DNA结合蛋白
原核表达
多克隆抗体
Human adenoviruses type 7
DNA-binding protein
Prokaryotic expression
Polyclonal antibody
