期刊论文

Expression of the classical swine fever virus E~(rns) in baculovirus expression system and the establishment of an indirect ELISA

中文

中国兽医学报

1005-4545

2020

40

03

450-456

10.16303/j.cnki.1005-4545.2020.03.02

湖南省教育厅一般项目（18C0165） 国家“十三五”重点研发计划资助项目（2017YFD0500102）

本校为第一机构

将人工合成的CSFV E~(rns)基因插入到pFastBac1获得重组转移载体pFastBac1-E~(rns),在DH10Bac大肠杆菌中转座后提取杆粒并转染Sf9细胞,用获得的重组杆状病毒感染High Five细胞表达E~(rns)蛋白并通过亲和层析进行纯化。以纯化后E~(rns)蛋白作为诊断抗原,通过摸索抗原包被量和抗体血清稀释倍数等条件,建立检测CSFV E~(rns)抗体的间接ELISA方法(E~(rns)-iELISA)。结果显示,E~(rns)蛋白得到有效表达,相对分子质量约为35 000;确定的ELISA抗原包被量为120ng/孔、血清稀释100倍,酶标二抗稀释6 000倍,封闭条件为37℃30min,TMB底物作用15 min,临界值为0.329。用E~(rns)-iELISA方法检测92份阴性血清,其特异...

Recombinant donor vector pFastBac-E~(rns) was obtained by inserting synthesized CSFV E~(rns) gene into pFastBac1,after transposition in DH10Bac E.coli,rBacmids were extracted and transfected into Sf9 cells.E~(rns) protein was expressed in High Five cells infected with the obtained recombinant baculovirus and purified by affinity chromatography.An indirect ELISA method was established using purified E~(rns) protein by exploring the concentration of coated antigen and dilution of serum samples.E~(rns) protein was expressed with a molecular weight...