【作者】 常巧呈；

【导师】 李太元； 金宁一；

【作者基本信息】 延边大学 ， 预防兽医学， 2008， 硕士

【摘要】 口蹄疫(Foot-and-Mouth disease,FMD)是由口蹄疫病毒(Foot-and-Mouthdisease virus,FMDV)引起偶蹄动物的最烈性传染病之一。目前FMD呈世界范围流行趋势。特别是近几年来O型、A型和AsiaⅠ型FMD在我国周边国家或东南亚地区时有发生,对我国养殖业构成了严重的威胁。以本室构建的复合多表位表达盒OAAT及AsiaⅠ型FMDV的P1-2A-3C为基础构建了FMDV核酸疫苗pIRES-OAAT-P1-2A-3C。用限制性酶切对其进行了鉴定。该DNA疫苗转染HeLa细胞表达的蛋白在间接免疫荧光(IFA)检测中与O型FMDV多克隆血清和AsiaⅠ型FMDV多克隆血清均能结合,在FITC标记的二抗作用下经伊文斯兰染色可见较好的绿色荧光。以BALB/c小鼠为试验动物,应用该DNA疫苗对小鼠进行免疫接种后,检测体液与细胞免疫指标。研究结果表明,pIRES-OAAT-P1-2A-3C及灭活苗免疫组的小鼠均能产生特异性抗体(与对照组相比,p＜0.05)。pIRES-OAAT-P1-2A-3C免疫组抗AsiaⅠ、O抗体效价,低于灭活疫苗免疫组。但均高于空质粒pIRElneo对照组及PBS对照组(p＜0.01)。流式细胞仪检测所得数据统计学分析结果表明,与灭活苗、空质粒pIRElneo对照组及PBS对照组比较,用所构建的DNA重组体DNA疫苗免疫的试验组小鼠的T淋巴细胞亚类CD4+和CD8+的数量显著提高(p＜0.05)。而各对照组之间差异不显著。用ELISPOT检测小鼠脾淋巴细胞分泌IFN-γ斑点数试验结果表明,pIRES-OAAT-P1-2A-3C免疫组IFN-γ斑点数量(64.726)较灭活苗(39.625)显著增多(p＜0.05)。而各对照组之间差异不显著。应用MTT法检测淋巴细胞增殖结果显示,pIRES-OAAT-P1-2A-3C和灭活苗组小鼠T淋巴细胞均出现较明显的特异性和非特异性增殖反应。且pIRES-OAAT-P1-2A-3C组增殖能力强于灭活疫苗组(p＜0.05)。给豚鼠进行2次免疫,用250ID50的FMDV进行攻击,观察其保护效果。灭活疫苗组4只豚鼠均未出现任何FMD症状,表明获得完全保护。pIRES-OAAT-P1-2A-3C组有2只获得完全保护。说明该疫苗具有一定保护效果。上述结果证实了表达盒OAAT与P1-2A-3C的表达组合在鼠体内的良好的免疫原性。为其它载体应用该组合进行疫苗研制提供了试验依据。为研制有效的口蹄疫多价苗的作了一定的试验准备并提供物质储备。更多还原

【Abstract】 Foot-and-mouth disease(FMD) caused by FMD virus(FMDV) is one of the most contagious animal virus diseases.At present,FMD is spreaded in all of the world.Serotype O,A and Asia I FMD once happened in history of China,and are often epidemic in the country around China and Southeast Asia.Our country has been threatened by Serotype O,A and Asia I FMD at present.The multivalent DNA vaccine pIRES-OAAT-P1-2A-3C was constructed using the cassette OAAT and P1-2A-3C(AsiaI),the recombinant plasmid was Identificated by restricted endonuclease。Transfect HeLa cells with the constructed multivalent FMDV DNA vaccine pIRES-OAAT-P1-2A-3C and identify the expressed product by IFA,and the result show that the production of expression could both react between FMDV polyclonal rabbit antibody of O type and FMDV polyclonal rabbit antibody of AsiaⅠtype.BALB/c mice which was used as experimental model were innoculated by DNA recombinant strain as before.The humoral and cell immunity level were detected.The results showed that special antibody aiming at O、AsiaⅠtype could be generated in group of pIRES-OAAT-P1-2A-3C.were lower than that of inactivated vaccination group,but is all higher than that of control group(p＜0.01).The result from flow cytometry after statistics analyse indicated that contrast with the inactivated vaccination group pIRE1neo group and PBS group the mice which immuned with pIRES-OAAT-P1-2A-3C,could found the significant elevation of T lympholeukocyte subgroup CD4+ and CD8+.The results of ELISPOT detection for number of IFN-γblot showed the number(64.726) of groups pIRES-OAAT-P1-2A-3C was higher (p＜0.05) than inactivated vaccination group(39.625).And the difference between control group were quiet.The results of Lymphoproliferation of peripheral blood lymphocytes showed that group of pIRES-OAAT-P1-2A-3C and inactivated vaccination group both to appear specificity and non-specificity Lymphoproliferation. Furthermore reproductive activity of pIRES-OAAT-P1-2A-3C group is better than inactivated vaccination group(p＜0.05).In this study,guinea pigs were inoculated intramuscularly twice.All of Guinea pigs were challenged with 250ID50 FMDV.To evaluate the prophylaxtic efficacy of this vaccine candidate,The four guinea pigs in inactivated vaccination group all not emerge FMD symptom.Indicate that the group has obtained completely protection. There is two guinea pigs obtained protected completely from the FMDV in the DNA vaccine group.The result show that the combination of cassette OAAT and P1-2A-3C(AsiaI) has good immunogenicity.It will give experiment base to develop vaccine using the combination for other vector..Also do certain preparation and provide material reserve for developing effective FMD multivalent DNA vaccine.更多还原