人巨细胞病毒pp65 IgG亲和指数在人巨细胞病毒原发感染小鼠模型中的标志作用  

The effect of the human cytomegalovirus pp65 IgG-avidity index for the human cytomegalovirus primary infection in mice model

作　　者：刘倩[1] 王明丽[1] 

机构地区：[1]安徽医科大学微生物学教研室 ,合肥230032

出　　处：《中华检验医学杂志》2008年第5期557-561,共5页Chinese Journal of Laboratory Medicine

基　　金：安徽省“十五”生物医药重大科技专项资助项目（01303003）

摘　　要：目的通过对人巨细胞病毒（human cytomegalovirus，HCMV）原发感染BALB／c模型小鼠感染状态的实验研究，探索检测HCMVpp65IgG亲和指数（avidityindex，AI）在原发感染诊断中的意义和作用。方法取6～8周龄SPF（SpecificPathogenFree）级BALB／c雌性小鼠30只，分为5组，每组6只，分别用2×10^6、2×10^5、2×10^4、2×10^3和2×10^2PFU／ml的5个剂量的病毒悬液腹腔注射小鼠，1．0mE／只；另取浓度为2×10^6PFU／ml剂量的病毒，经56℃30min灭活后，1．0ml／只腹腔注射雌性小鼠共6只，作为灭活对照组；同株同代的HF细胞悬液1．0ml／只腹腔注射雌性小鼠共6只，作为HF细胞对照组。各组小鼠于屏障系统内饲养1个月后，检测小鼠的感染状态。病毒分离检测脑、肺组织中感染性病毒颗粒，观察HCMV特异性细胞病变效应，PCR试验检测细胞培养物UL83基因，间接免疫荧光试验检测细胞玻片pp65抗原；逆转录-聚合酶链反应（RT-PCR）检测小鼠脑、肺组织中HCMVpp67的转录；同时，用本室自制的截短pp65抗原制备的ELISA试剂盒检测血清标本中HCMVpp65特异性IgM抗体和IgG．AI。结果2×10^6PFU／ml和2×10^5PFU／ml剂量的病毒感染小鼠后，脑、肺组织中均可检测到感染性病毒颗粒，感染率均为100％；RT-PCR均可检测到小鼠脑、肺组织中pp67转录产物；血清学检测这两组小鼠HCMVpp65IgM均为阳性，HCMVpp65IgG—AI〈50％；检测结果判断该两组小鼠为HCMV原发感染。余低剂量病毒组、灭活病毒组和人胚成纤维细胞（HF）对照组检测结果均为阴性。结论HCMVAD160株可感染BALB／c小鼠，初次感染病毒1个月后可表现原发感染状态；以HCMVpp65重组蛋白为抗原检测特异性kM抗体以及相应ZgG-AI间接ELISA法，可作为对HCMV原发感染的初筛手段，是诊断HCMV原发感染有效的方法之一。Objective To investigate the significance of human cytomegalovirus（HCMV） pp65 IgG antibody avidity index （AI） for the clinical diagnosis of HCMV primary infection through the experimental model of HCMV primary infection in BALB/c mice. Methods 6 - 8 weeks, female, specific-pathogen-free BALB/c mice were divided into 5 groups, 6 mice in each group. And the mice were injected with 2 ×10^6 PFU/ml, 2 ×10^5 PFU/ml, 2 ×10^4 PFU/ml, 2 ×10^3 PFU/ml and 2 ×10^2 PFU/ml of HCMV intraperitoneally respectively. Another 6 mice were injected intraperitoneally with the maximum dose of HCMV kept at 56℃ for 30 min as inactivated virus group, and HF negative control group was established at same time. All the mice were sacrificed to obtain brain and lung tissues for the following experiments after 1 month. （1） Tissue samples obtained from mice were inoculated in human embryo fibroblasts （HF） monolayers after routine treatment for virus isolation. HCMV specific cytopathic effect （CPE） was observed by inverted phase-contrast microscopy. HCMV UL83 DNA in the cultures was tested by PCR and pp65 antigen was detected by indirect immunofluorescence. （2） Extracted mRNA from tissue samples and HCMV pp67 mRNA were analyzed by reverse transcriptase PCR（RT-PCR）. （3）Immunoglobulin M （IgM） antibody and immunoglobulin G （IgG） antibody avidity was investigated for their usefulness in distinguishing primary genital HCMV infections from nonprimary infections with ELISA kit using truncated pp65 protein. Results HCMV can be isolated in the tissues from the mice injected with 2 ×10^6 PFU/ml and 2 ×10^5 PFU/ml. RT-PCR and ELISA showed positive results in the same groups. The infective rates were 100%. The analysis of the low doses groups, inactivated group and HF negative control group all showed negative results. Conclusions BALB/c mice can be infected with HCMV and appeared as primary infection after 1 month. Determination of HCMV pp65 IgM and HCMV pp65 IgG-AI by ELISA incorporated with virus isolation

关 键 词：巨细胞病毒感染 疾病模型 动物 磷蛋白类 免疫球蛋白G 抗体亲和力 酶联免疫吸附测定 

分 类 号：R686[医药卫生—骨科学]