人巨细胞病毒流行病学调查及其核酸疫苗研究

【作者】 马道新；

【导师】 于修平；

【作者基本信息】 山东大学 ， 病原生物学， 2004， 博士

【摘要】 目的 应用流行病学和分子生物学等方法,对山东省HCMV感染情况及其相关影响因素进行深入研究,并探讨其发生规律及相互关联性,为进一步进行HCMV的疫苗研制及应用提供事实和理论依据。 方法 利用流行病学调查方法,随机抽取山东省19个县区的1870份血样,并进行相关资料收集;利用ELISA方法进行HCMV IgM和IgG抗体的检测,同时进行ABO血型的检测,利用PCR-RFLP方法对感染相关基因SDF1受体和CCR2受体基因的多态性进行检测;利用Microsoft Excel和SAS统计学软件对山东省HCMV IgM和IgG阳性率进行统计学分析,同时研究其与地区、性别、职业、年龄、血型以及SDF1和CCR2受体基因多态性等相关因素之间的关系。 结果 山东省HCMV IgM阳性率为40.21%,IgG阳性率为48.07%;各地区间IgM和IgG阳性率有显著性差异;女性IgM和IgG显著高于男性;农民和医生IgG显著高于其它职业;随着年龄的增加IgG阳性率增加;HCMV感染与血型、SDF1和CCR2受体基因多态性等因素无显著性关联。 结论 山东省HCMV既往和现行感染率都较高,与地区、性别、职业、年龄等因素相关,与血型、SDF1和CCR2受体基因多态性无关。更多还原

【Abstract】 Objective To study HCMV infection rate of Shandong province and its related factors using epidemiology and molecular biology methods. To explore its occurrence rule in Shandong province and provide base for HCMV vaccine study.Methods 1870 blood samples of Shandong province and their related information were collected randomly using epidemiological methods. HCMV IgM and IgG antibodies of these samples were detected using ELISA method. ABO blood types were also detected. SDF1 receptor and CCR2 receptor genes polymorphisms were detected using PCR-RFLP method. Microsoft Excel and SAS statistical softwares were used to study HCMV IgM and IgG positive rates and their relationships with sex, age, work, blood type, and SDF1 and CCR2 receptor genes polymorphisms.Results Shandong province positive rate of HCMV IgM was 40.21%, while that of IgG was 48.07%. There were significant differences of IgM and IgG positive rates between districts. The positive rates of IgM and IgG for female were higher than those for male. The IgG positive rates of peasants and doctors were higher than those of other works. The IgG positive rate was increasing while the age became older. There were no significant associations between HCMV and blood types, SDF1 and CCR2 receptor genes polymorphisms.Conclusions The HCMV positive rate at present was high, and it was correlated with geography, sex, work and age, but it was not associated with blood type and SDF1 and CCR2 receptor genes polymorphisms. Objective To construct the HCMV phosphoprotein 65 (pp65) expression vector using molecular clone methods and to explore its in vivo immunological effects.Methods HCMV pp65 fragment was cut down from pCMVintpp65 vector and inserted into pcDNA3 vector to construct pp65 expression vector pcDNA3.pp65. pcDNA3.pp65 vector was amplified after being certificated by enzymation with BamH I. It was immunized into BALB/C mice as DNA vaccine through muscle and hypodermic approaches with three doses, and immunization was enhanced at the second, fourth and eighth weeks. The mice weight and temperature were measured at the second, fourth, eighth and tenth weeks after immunization. The T cell proliferation activity and NK activity were determined using MTT method. The sera IgM and IgG levels of HCMV were detected using ELISA method. The mixture of immunized sera and diluted virus was used to infect human embryonic lung fibroblast cells to detect its virus neutralization.Results The expression vector pcDNA3.pp65 was constructed successfully and was proved correct after enzymation with BamH I. There was no apparent difference of weight and temperature between the immunized mice groups. The T cell proliferation activity, NK cell activity, HCMV IgM and IgG antibodies were increased with the time and dose. It appeared that the immunized sera could neutralize HCMV infection, but there was no apparent difference between the control and the experimental mice.Conclusion HCMV DNA vaccine pcDNA3.pp65 could induce apparent cellular and humoral immunological reaction. It may be used as a candidate vaccine to protect human from HCMV infection.Objective To construct the HCMV DNA vaccine vectors pCMV.gB, pCMV.gB.pp65, pVP22.pp65, pVP22.gB and pVP22.gB.pp65. To evaluate and compare their in vivo immunological effects after being immunized into mice.Methods 1. Construction of DNA vaccine vectors: (1). Construction of pVP22.gB: gB gene fragment was amplified from pCAGGS.gB plasmid using PCR method and inserted into pVP22/myc-His-TOPO TA vector; (2). Construction of pVP22.gB.pp65: pVP22.gB plasmid was enzymed with Kpn I and BamH I, and pp65 fragment was obtained when pCMVintpp65 was also enzymed with Kpn I and BamH I. pp65 fragment was inserted into pVP22.gB using T4 ligation enzyme; (3). Construction of pVP22.pp65: pVP22.pp65 was constructed when gB fragment was cut off from pVP22.gB.pp65 using Xba I and BamH I; (4). Construction of pCMV.gB: pCMV.gB was constructed when VP22 gene was cut off from pVP22.gB using Hind III and Kpn I ; (5). Construction of pCMV.gB.pp65: pp65 fragment was inserted into pCMV.gB when enzymed with BamH I. 2. Immunological evaluation of HCMV DNA vaccines: 7 groups of BALB/C mice (pcDNA3.pp65, pCMV.gB, pCMV.gB.pp65, pVP22.pp65, pVP22.gB, pVP22.gB.pp65 and control ) were immunized s.c into BALB/C mice at week 0, 2, 4, 8. MTT method was used to detect T cell proliferation activity and IL-2 biological activity. LDH release test was used to detect NK activity. The sera IgM and IgG levels of HCMV were detected using ELISA method. The更多还原