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。
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.
作者
严杰聪
王帅勇
王曼茱
王娟
荣新利
邢燕茹
虞凌雪
周艳君
单同领
童武
郑浩
刘长龙
童光志
于海
YAN Jiecong;WANG Shuaiyong;WANG Manzhu;WANG Juan;RONG Xinli;XING Yanru;YU Lingxue;ZHOU Yanjun;SHAN Tongling;TONG Wu;ZHENG Hao;LIU Changlong;TONG Guangzhi;YU Hai(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第4期79-84,共6页
Chinese Journal of Animal Infectious Diseases
基金
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。
关键词
猪圆环病毒2型
CAP蛋白
同源重组
表达纯化
ELISA
Porcine circovirus type 2
Cap protein
homologous recombination
protein expression and purification
ELISA
