基于抗体融合荧光蛋白的镰刀菌免疫检测技术
Immunological Detection Technology for Fusarium Based on Antibody Fusion Fluorescent Proteins
闫瑜雪 1尚国富 2陈绍美 3喻艳琴 2胡祖权 4艾民 5彭晓燕3
作者信息
- 1. 贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳,550000
- 2. 贵州医科大学基础医学院,贵州省感染免疫与抗体工程特色重点实验室,贵阳,550000
- 3. 贵州医科大学生物与工程学院(健康医药现代产业学院),贵州省高等学校免疫细胞与抗体工程研究中心,贵阳,550000
- 4. 贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳,550000;贵州医科大学基础医学院,贵州省感染免疫与抗体工程特色重点实验室,贵阳,550000;贵州医科大学生物与工程学院(健康医药现代产业学院),贵州省高等学校免疫细胞与抗体工程研究中心,贵阳,550000
- 5. 贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵阳,550000;贵州医科大学生物与工程学院(健康医药现代产业学院),贵州省高等学校免疫细胞与抗体工程研究中心,贵阳,550000
- 折叠
摘要
镰刀菌(Fusariam)是一类重要的植物病原菌和临床上常见的致病真菌,为实现镰刀菌的快速灵敏检测,本研究通过基因工程操作技术将编码抗镰刀菌单链抗体(single-chain variable fragment,scFv)FvSG7的基因与mNenoGreen(mNG)绿色荧光蛋白和NanoLuc(NLuc)荧光素酶基因构建融合表达载体,优化原核表达条件后进行大量表达纯化;利用SDS-PAGE和Western blot检测融合蛋白FvSG7-mNG-NL的表达情况;利用免疫荧光分析融合蛋白与镰刀菌菌丝的结合特性;利用酶联免疫吸附测定实验(enzyme-linked immunosorbent assay,ELISA)分析融合蛋白与镰刀菌抗原的结合能力.结果显示,融合荧光蛋白FvSG7-mNG-NL的原核表达载体构建成功,并实现可溶性高效表达.FvSG7-mNG-NL保留单链抗体、荧光素酶以及荧光蛋白的活性,可通过生物发光共振能量转移(bioluminescence resonance energy transfer,BRET)增强发光成像.本研究为发展镰刀菌的灵敏快速免疫检测技术奠定了实验基础.
Abstract
Fusarium is an important class of plant pathogens and common pathogenic fungi in clinical practice.In order to achieve rapid and sensitive detection of Fusarium,fusion expression vectors containing the coding gene of the anti-Fusarium single-chain variable frag-ment(scFv)antibody FvSG7,mNenoGreen(mNG)green fluorescent protein and NanoLuc(NLuc)luciferase was constructed by genetic engineering techniques.After optimizing the prokaryotic expression conditions,a large amount of fusion protein was expressed and puri-fied.The expression of the FvSG7-mNG-NL fusion protein was detected by using SDS-PAGE and Western blot.And their binding proper-ties to the mycelia of Fusarium was analyzed using immunofluorescence.Enzyme-linked immunosorbent assay(ELISA)was performed to compare the binding capacity of fusion protein with the antigen of Fusarium.The results showed that the prokaryotic expression vector of the FvSG7-mNG-NL fusion fluorescent protein was successfully constructed and the soluble and efficient expression was achieved.The FvSG7-mNG-NL fusion retained the activities of scFv antibody,luciferase and fluorescent protein,and could enhance luminescence ima-ging through bioluminescent resonance energy transfer(BRET).Therefore,this study lays a practical foundation for the development of sensitive and rapid immunological detection technology for Fusarium pathogens.
关键词
镰刀菌/单链抗体/荧光素酶/融合蛋白/免疫学检测Key words
Fusarium/Single-chain variable fragment(scFv)/Luciferase/Fusion protein/Immunological detection引用本文复制引用
出版年
2024
NETL
NSTL
万方数据