肠道病毒遗传性分析及早期实验室诊断方法的研究

发布时间：2018-01-12 03:24

本文关键词：肠道病毒遗传性分析及早期实验室诊断方法的研究　出处：《福建医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：肠道病毒型别多,变异大,是多种急慢性疾病的主要病原。在脊髓灰质炎病毒消灭后期,本研究以福建省57 例急性弛缓性麻痹(AFP)病例中分离到的非脊髓灰质炎肠道病毒(non-polio enterovirus,NPEV)为对象,用传统血清中和试验结合分子生物学技术进行诊断和定型。结果显示,应用组合血清进行中和试验定型,54.39%病毒能定出型别,45.61%无法定型。而应用分子生物学技术不仅鉴定了其型别,发现两株新型病毒FJ96-71 和FJ02-47,还及时快速地对一起病毒性脑炎流行的病原进行了鉴定与分析。可见分子生物学鉴定方法较传统方法不仅省时快速,而且能对传统方法所无法定型的病毒进行鉴定,及时地确定疾病流行的病原。 FJ96-71 病毒经蚀斑纯化后,通过全基因组序列测定与分析,结合电镜形态学诊断、细胞培养时病变特点和抗原性分析,确认属于肠道病毒新血清型HEV75,并制备了型特异性中和抗体,为今后相应疾病的预防控制作准备。福建省NPEV 流行型别丰富,其中不仅含国外报道较多与无菌性脑炎等严重后果疾病关系密切型别—ECV6、ECV11、ECV13 和ECV30 等,而且包含最近国外未见报道却引起本地无菌性脑炎较大范围流行的型别—ECV19。对肠道病毒抗原位点集中且与血清型相关性好的衣壳蛋白VP1 基因全长或部分序列进行遗传性分析,结果显示,肠道病毒福建分离株与国外参考株间差异大,具本土特点。部分血清型分离株间也存在较大差异,说明一个血清型在福建省内还存在多传播链流行。因此遗传性分析在追踪病毒性疾病流行来源具有重要意义。借鉴国外对肠道病毒通用抗原研究的成果,结合福建分离株遗传特征,以福建株为模板,体外扩增通用抗原目的基因片段,利用基因工程重组技术,用pET30a 和pET42a 表达系统在大肠杆菌BL21 中进行表达,诱导后重组蛋白表达量约占总体蛋白表达量的40%。经Western blot 检测,重组蛋白能被国外商品化的肠道病毒组特异性的5-D8/1 单抗、含PV 抗体的人血清与分别代表肠道病毒A、B、D 三组的单型血清所识别。制备其相应的多抗和单抗,与5-D8/1 单抗同时应
[Abstract]:Enterovirus is the main pathogen of many acute and chronic diseases. It is the main pathogen of many acute and chronic diseases. Non-poliovirus non-polio enterovirus was isolated from 57 cases of acute flaccid paralysis (AFP) in Fujian Province. NPEV) was used for diagnosis and typing by traditional serum neutralization test and molecular biological technique. The results showed that 54.39% of the viruses could be identified by using combined serum neutralization test. 45.61% could not be typed. The molecular biology technology not only identified its type, but also found two new viruses FJ96-71 and FJ02-47. The identification and analysis of a viral encephalitis pathogen was also carried out in time and quickly. It can be seen that molecular biological identification method is not only faster than the traditional methods. And it can identify the virus which the traditional method can not be stereotyped, and identify the pathogen of the disease in time. After the FJ96-71 virus was purified by plaque, the whole genome sequence was determined and analyzed, combined with the morphological diagnosis of electron microscope, the pathological characteristics and antigenicity of cell culture were analyzed. The new serotype HEV75 of enterovirus was confirmed and the type-specific neutralizing antibody was prepared to prepare for the prevention and control of the disease in the future. Fujian Province is rich in epidemic types of NPEV, which not only contains more foreign reports and aseptic encephalitis and other serious consequences of diseases closely related to the type-ECV6 ECV11. ECV13, ECV30 and so on, but not recently reported abroad but caused by local aseptic encephalitis in a wide range of types-ECV19. The full-length or partial sequence of capsid protein VP1 gene which has concentrated antigen sites of enterovirus and has good correlation with serotype was analyzed. The difference between Fujian isolate and foreign reference strain of enterovirus is great, and there are some differences among some serotype isolates. It is suggested that a serotype still exists in Fujian province, so genetic analysis is of great significance in tracing the source of viral diseases. Referring to the achievements of foreign research on common antigen of enterovirus, combining with the genetic characteristics of Fujian isolate, using Fujian strain as template, the target gene fragment of common antigen was amplified in vitro, and the recombinant technology of gene engineering was used. E. coli BL21 was expressed by pET30a and pET42a expression system. After induction, the expression of recombinant protein accounted for about 40% of the total protein expression, which was detected by Western blot. The recombinant protein can be obtained by the foreign commercial enterovirus group specific 5-D8 / 1 monoclonal antibody, the human serum containing PV antibody and the representative of enterovirus Agna B, respectively. Group D is recognized by monotypic serotypes. The corresponding polyantibodies and McAbs are prepared, which should be matched with 5-D8 / 1 McAbs.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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