摘要
目的构建甲型HSN1禽流行性感冒流感病毒血凝素（HA）抗原和霍乱毒素B亚单位（CTB）融合蛋白真核表达载体，并研究其在COS7细胞中的表达特性，及其在动物体内不同时间点诱导机体产生特异性抗体的情况。方法采用PCR技术分别克隆CTB基因和HA基因，并用BamHI酶切2个基因片段，在T4连接酶的作用下构建CTHA融合基因（CH基因）。双酶切后，将CH基因插入真核表达质粒pCI—neo中，构建甲型H5N1禽流感病毒融合蛋白真核表达载体（pCI—CH）。将pCICH质粒转染COS7细胞，利用Western印迹、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SD孓PAGE）检测HA抗原的表达；间接EI-ISA法检测免疫接种新西兰大白兔后其血清中HA抗原的特异性抗体效价，以及与H1N1、H9N1、H3N2和乙型流感病毒的交叉反应情况。结果pCl—CH真核表达载体（即核酸疫苗）构建成功，可在cos7细胞中高效表达，并能在大白兔体内诱导产生特异性抗pcI-cH抗体。核酸疫苗pC-CH特异性抗血清不仅可以与甲型HSNl流感病毒特异性结合（P／N〉2．1），而且可以与HINl、HgNl和H3N2毒株发生特异性反应；但该抗血清与乙型流感病毒无特异性反应。结论构建的甲型H5N1禽流感病毒核酸疫苗具有良好的免疫原性。
Objective To construct a eukaryotic vector which contains avian H5N1 influenza virus hemagglutinin （HA） antigen and the cholera toxin B subunit （CTB） and to investigate its expression in COS7 cells, and the ability to induce specific immune responses in vivo in different periods. Methods After cloned by polymerase chain reaction （PCR）, CTB and HA genes were digested with BarnH I and connected into CTB-HA gene with T4 ligase. The connected gene was referred to as CH. After double digestion, CH gene was inserted into a eukaryotie recombinant plasmid pCI-neo. The pCI-CH plasmid was then transfected into COS7 cells. Western blot was used to detect the expression of HA antigen. After New Zealand white rabbits were immunized, the titer of HA antigen-specific antibody in serum and its specificity with other strains such as H1N1, H9N1, H3N2 and influenza B virus were determined by indirect enzyme-linked immunosorbent assay.Results The pCI-CH vector （DNA vaccine） was successfully constructed, which could be efficiently expressed in COS7 cells and induce specific antibodies against pCI-CH in rabbits. Cross reactions indicated that DNA vaccine pCI-CH specific antisera could not only react with HSN1 strain （P/N：〉 2.1）, but also H1N1, H9N1 and H3N2 strains, but did not cross react with influenza B virus. Conclusion The newly constructed avian H5N1 influenza virus nucleic acid vaccine has good immunogenieity.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2013年第7期386-391,共6页
Chinese Journal of Infectious Diseases
基金
武汉市卫生局科研项目
