CAJ | 학술논문

目的构建甲型H5N1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BamH Ⅰ酶切2个基因片段,在T4连接酶的作用下构建CT-HA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pCI-CH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(SDS-PAGE)检测HA抗原的表达；间接ELISA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 pCI-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pCI-CH特异性抗血清不仅可以与甲型H5N1流感病毒特异性结合(P/N＞2.1),而且可以与H1N1、H9N1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.
목적 구건갑형H5N1금류행성감모류감병독혈응소(HA)항원화곽란독소B아단위(CTB)융합단백진핵표체재체,병연구기재COS7세포중적표체특성,급기재동물체내불동시간점유도궤체산생특이성항체적정황.방법 채용PCR기술분별극륭CTB기인화HA기인,병용BamH Ⅰ매절2개기인편단,재T4련접매적작용하구건CT-HA융합기인(CH기인).쌍매절후,장CH기인삽입진핵표체질립pCI-neo중,구건갑형H5N1금류감병독융합단백진핵표체재체(pCI-CH).장pCI-CH질립전염COS7세포,이용Western인적、십이완기류산납취병희선알응효전영(SDS-PAGE)검측HA항원적표체；간접ELISA법검측면역접충신서란대백토후기혈청중HA항원적특이성항체효개,이급여H1N1、H9N1、H3N2화을형류감병독적교차반응정황.결과 pCI-CH진핵표체재체(즉핵산역묘)구건성공,가재COS7세포중고효표체,병능재대백토체내유도산생특이성항pCI-CH항체.핵산역묘pCI-CH특이성항혈청불부가이여갑형H5N1류감병독특이성결합(P/N＞2.1),이차가이여H1N1、H9N1화H3N2독주발생특이성반응；단해항혈청여을형류감병독무특이성반응.결론 구건적갑형H5N1금류감병독핵산역묘구유량호적면역원성.
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 BamH 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 eukaryotic 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 H5N1 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 immunogenicity.