摘要
为了建立丙型肝炎病毒抗体 (HCV Ab)的双抗原夹心ELISA检测方法 ,克隆表达带有生物素标签的HCV多种抗原优势表位融合蛋白 ,选取HCV各抗原如Core,NS3,NS4 ,NS5和E的优势抗原表位片段编码序列 ,克隆重组为融合基因 ,插入PinpointTM Xa 1T载体中诱导表达 ,并用Westernblot进行抗原性及标签生物素活性鉴定 ;表达抗原经PromegaSoftLinkSoftReleaseAvidinResin系统亲和层析纯化后包被酶联板 ,用抗HCV单片段抗体阳性血清对表达融合蛋白各区的抗原性作间接ELISA法鉴定。结果显示 :成功构建了带有生物素标签的HCV多抗原表位融合基因表达载体 ,该载体可在JM10 9(DE3)中可溶性表达目的蛋白 ,表达产物携带生物素标签 ,融合的各片段区均具有良好的抗原性。结论 :所构建融合抗原可可溶性表达 ,可以用作双抗原夹心ELISA的酶标抗原 ,所含生物素标签也可用作酶联检测的生物素—亲和素信号放大系统。
The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core,E1,E2,NS3,NS4,and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin,the protein was pre-coated on microwell and detected with anti-core,anti-NS3,anti-NS4 and anti-NS5 positive sera by EIA,respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully,it reacted with anti-HCV positive serum,and exposed all of the major immunogenic epitopes chosen. In conclusion,this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.
出处
《中国实验血液学杂志》
CAS
CSCD
2004年第3期359-362,共4页
Journal of Experimental Hematology
关键词
丙型肝炎病毒
融合抗原
ELISA
生物素
hepatitis C virus
fusion antigen
enzyme linked immunosorbent assay
biotin
