双亚型(H5/H7)流感病毒DNA疫苗的构建及其免疫原性检测  

Construction of dual-subtype (H5/H7) influenza virus DNA vaccine and determination of its immunogenicity

作　　者：白靓[1] 金宁一[2] 成岩[1] 石毅[1] 鲁会军[2] 田明尧[2] 南文龙[2] 赵翠青[2] 张金双[2] 关铭[1] 

机构地区：[1]吉林大学药学院药物分析教研室,吉林长春130021 [2]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《吉林大学学报（医学版）》2010年第2期267-270,I0002,共5页Journal of Jilin University：Medicine Edition

基　　金：国家高技术研究发展计划(863计划)资助课题(2006AA10A205)

摘　　要：目的:构建双亚型(H5/H7)流感病毒DNA疫苗,并检测其免疫原性。方法:构建共表达流感病毒H5亚型血凝素(HA)和H7亚型HA的DNA疫苗,采用间接免疫荧光法(IFA)检测目的蛋白在幼仓鼠肾细胞(BHKcell)中的表达。分为pVAX1-H5-H7、pVAX1-H5、pVAX1-H7和pVAX1空质粒对照组4组(每组15只)进行小鼠免疫实验,并应用ELISA法检测小鼠血清抗体,流式细胞术检测小鼠脾T淋巴细胞亚群数量。结果:所构建的双亚型(H5/H7)流感病毒DNA疫苗在BHK细胞中的表达产物可激发荧光抗体产生绿色免疫荧光,且对照pVAX1空载体转染BHK细胞无免疫荧光产生。使用双亚型(H5/H7)流感病毒DNA疫苗免疫小鼠后,小鼠产生了针对H5和H7抗原的血清特异性抗体,流式细胞术检测免疫组小鼠脾T淋巴细胞亚群CD4+和CD8+的数量显著高于对照组(P<0.05)。结论:成功构建的双亚型(H5/H7)流感病毒DNA疫苗可以诱导小鼠产生特异性的细胞免疫和体液免疫应答。Objective To construct the dual-subtype（H5/H7） influenza virus DNA vaccine and determine its immunogenicity.Methods Influenza virus DNA vaccine pVAX1-H5HA-H7HA co-expressing H5 subtype hemagglutinin（HA） and H7 subtype HA was constructed.The expressed target proteins in BHK cells were identified by IFA.Immunized mice were divided into four groups（15 mice in each group）：pVAX1-H5-H7,pVAX1-H5,pVAX1-H7 and pVAX1 control groups,and the serum antibodies were detected by ELISA,the number of splenic T lymphocyte subgroups was determined by flow cytometry.Results The product of constructed dual-subtype（H5/H7） influenza virus DNA vaccine in BHK cells stimulated the expression of green immunofluorescence of fluorescent antibody,and the pVAX1 empty vector transfected BHK cells had no immunofluorescence.The immunized mice produced the serum specific antibodies.The number of splenic T lymphocyte subgroups CD4＋ and CD8＋ was higher than that in control group（P〈0.05）.Conclusion Dual-subtype（H5/H7） influenza virus DNA vaccine is successfully constructed and this DNA vaccine can induce specific cellular and humoral immune responses in mice.

关 键 词：流感病毒A型 疫苗 DNA 体液免疫 细胞免疫 

分 类 号：Q81[生物学—生物工程]