中国兽医科学2017，Vol.47Issue(10)：1221-1226,6.DOI:10.16656/j.issn.1673-4696.2017.10.003
马流感病毒NP基因的原核表达及其间接ELISA检测方法的建立
Prokaryotic expression of equine influenza virus NP gene and establishment of indirect ELISA detection method
摘要
Abstract
The NPgene was amplified from equine influenza virus(EIV) of A/equine/Heilongjiang/ SS1/2013(H3N8) with RT-PCR,and then cloned into pET-32a(+) to construct the expression plasmid pET-NP.After sequencing,the correct plasmid was transformed into E.coki Rosetta(DE3),induced by IPTG,and purified by Ni-NTA purification system to obtain the recombinant NP (rNP).To establish a rapid method to detect EIV antibodies in the equine sera,an indirect ELISA assay was developed with rNPas coating antigen.The results showed that under the condition of 25 ℃ and 0.2 mmol/L IPTG,the recombinant protein had the highest expression level and was soluble in the supernatant.The purified rNPhad no any cross reactions with positive sera against equine rhinopeumonitis virus,equine infectious anemia virus,and R.equi.The establ;shed detection method had a higher sensitivity than hemagglutination inhibition test.In addition,the coefficients of variations in both inter and intra assay were less than 15％.The established assay can provide an effective method for the serological test of equine influenza in China.关键词
马流感病毒/NP基因/原核表达/间接ELISAKey words
equine influenza virus/NP gene/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
何栋,卢刚,罗霭剑,孙凌霜,贾坤,沈丹,李守军..马流感病毒NP基因的原核表达及其间接ELISA检测方法的建立[J].中国兽医科学,2017,47(10):1221-1226,6.基金项目
国家自然科学基金项目(31402259) （31402259）
广东省兽医临床重大疾病综合防控重点实验室项目(2013A061401013) （2013A061401013）
