猪圆环病毒2型Cap蛋白的原核表达及间接ELISA抗体检测方法的初步建立
Prokaryotic Expression of Porcine Circovirus Type 2 Capsid Protein and Preliminary Establishment of Indirect ELISA Antibody Detection Method
严杰聪 1王帅勇 1王曼茱 1王娟 1荣新利 1邢燕茹 1虞凌雪 1周艳君 1单同领 1童武 1郑浩 1刘长龙 1童光志 1于海2
作者信息
- 1. 中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241;江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州 225009
- 折叠
摘要
Cap蛋白作为猪圆环病毒2型(PCV2)的主要结构蛋白,构成病毒的核衣壳,是PCV2的主要免疫保护性抗原,在PCV2血清学诊断中具有重要意义.本研究根据PCV2的ORF2基因序列设计特异性引物,通过PCR方法扩增得到去核定位信号肽的ORF2基因,并通过同源重组将其克隆至原核表达载体pCold-Ⅰ中,经测序鉴定成功获得重组质粒 pCold-Ⅰ-Cap.将重组质粒转化至感受态细胞BL21中进行表达,通过考马斯亮蓝染色以及免疫印迹试验检测Cap蛋白的表达,同时将纯化后的重组蛋白通过优化反应条件建立检测PCV2血清抗体的间接ELISA方法.结果表明:重组去核定位信号肽Cap蛋白可溶性表达且可以与PCV2阳性血清特异性结合,通过探索不同蛋白包被浓度以及不同一抗孵育浓度初步建立了间接ELISA检测方法,进而为PCV2抗体的有效监测奠定基础.
Abstract
Cap protein,as the main structural protein of Porcine circovirus type 2(PCV2),constitutes the nucleocapsid of the virus and is the main immune protective antigen of PCV2,which has important significance in the serological diagnosis of PCV2.In this study,specific primers were designed according to the ORF2 gene sequence of PCV2,and the ORF2 gene of nuclear de-localization signal peptide was amplified by PCR,and cloned into the prokaryotic expression vector pCold-Ⅰthrough homologous recombination.The recombinant plasmid pCold-Ⅰ-Cap was successfully obtained after sequencing identification.The recombinant plasmid was transformed into the competent cell BL21 for expression.The expression of Cap protein was detected by Coomassie bright blue staining and Western blot assay.At the same time,an indirect ELISA method was established to detect PCV2 serum antibodies by optimizing reaction conditions.The results showed that the recombinant de-localization signal peptide Cap protein was soluble and could specifically bind to PCV2 positive serum.An indirect ELISA method was preliminarily established by exploring different protein envelope concentrations and different primary antibody incubation concentrations,thus laying a foundation for effective monitoring of PCV2 antibodies.
关键词
猪圆环病毒2型/Cap蛋白/同源重组/表达纯化/ELISAKey words
Porcine circovirus type 2/Cap protein/homologous recombination/protein expression and purification/ELISA引用本文复制引用
基金项目
国家自然科学基金面上项目(31970175)
国家生猪现代产业技术体系建设项目(NYCYTX-009)
中国农业科学院创新工程"猪病毒性繁殖障碍综合症团队"项目()
出版年
2024
国家科技期刊平台
NSTL
万方数据