我国人博卡病毒的分子流行病学及衣壳蛋白独有区表达纯化的研究
发布时间：2018-09-07 11:42
【摘要】： 在儿童和少年中，90％以上的上呼吸道感染是由病毒引起的。引起感冒的病毒种类很多，至少涉及7个病毒科的10多类共200多个型别的病毒。Heikkinen T，Jarvinen A(2003)认为引起呼吸道感染的病毒还有约1／4尚未被发现。近年来，呼吸道病毒频繁爆发或局部流行，旧的病毒不断变异，新的病毒不断产生。2001年，荷兰学者首次发现一种新的呼吸道病毒—人偏肺病毒；2003年SRAS爆发；继SARS爆发后，又有两种新的冠状病毒分别被发现：荷兰学者报道的人冠状病毒NL63(Coronavirus NL63)及香港首次发现的人冠状病毒HKU1(Coronavirus HKU1)；2005年瑞典学者报道一种新的细小病毒—人博卡病毒(Human Bocavirus，HBoV)，同样与儿童呼吸道感染有关，已受到广泛关注。目前世界各国对HBoV的研究主要集中于检测方法的建立和HBoV感染的流行病学调查。本课题主要研究我国人博卡病毒(Human Bocavirus，HBoV)的分子流行病学，在实验室初步建立HBoV的分子生物学检测方法和血清学诊断方法。主要目标是希望从分子水平来阐明我国HBoV病毒的流行、分布、变异及与疾病的相关性，并通过抗原蛋白的表达纯化，为建立血清学诊断方法研究奠定基础，并可为进一步研究病毒蛋白的功能和病毒相关疾病的治疗提供参考，为HBoV可能的病原检测监测提供科学依据。 一、HBoV分子流行病学 1、HBoV的分子检测 1)从因呼吸道感染而住院的儿童的鼻咽抽吸物样本中提取总核酸，逆转录后，PCR扩增目标病毒基因保守区，如凝胶电泳显示该片段并测序证实，则可确定为目标病毒阳性。 2)从因呼吸道感染而住院的儿童的鼻咽抽吸物样本中提取总核酸，逆转录。选择人博卡病毒的NS1基因作为目标基因，设计荧光定量PCR引物和检测探针，以重组质粒为标准品建立标准曲线，从而建立特异性检测人博卡病毒的荧光定量PCR方法。 2、HBoV流行病学调查 调查包括HBoV在特定人群中的发生、发展和疾病及健康有关状态与分布规律。结果显示HBoV主要与支气管炎，肺炎和支气管肺炎相关联，，在所检测的人群中的发生率约为8.3％，患者主要为2岁以下的婴幼儿。 3、HBoV基因的克隆及衣壳变异分析 利用克隆的HBoV基因和Genbank中的数据分析HBoV的变异规律。 二、VP1-U蛋白表达纯化 分析HBoV衣壳蛋白(VP1)序列抗原性，选择抗原性较高的VP1独有区(VP1-U)表达。将VP1-U的核酸序列进行密码子亲嗜改造，插入到pET30a(+)中(3’末端带His标签)，转化大肠杆菌BL21(DE3)。IPTG诱导表达，超声裂解细菌，镍柱亲和层析获得纯品。
[Abstract]:More than 90% of upper respiratory infections in children and adolescents are caused by the virus. There are many kinds of viruses that cause colds, at least 10 kinds of viruses of more than 200 types in seven viridae. Heikkinen Tu Jarvinen A (2003) thinks that about a quarter of the viruses that cause respiratory tract infections have not yet been found. In recent years, respiratory viruses have frequently erupted or become endemic, the old viruses have mutated and new viruses have been produced. In 2001, Dutch scholars first discovered a new respiratory virus, human metapneumovirus; SRAS outbreak in 2003; and following the outbreak of SARS. Two new coronaviruses were found: human coronavirus (Coronavirus NL63) reported by Dutch scholars and HKU1 (Coronavirus HKU1) first discovered in Hong Kong; In 2005, Swedish scholars reported a new parvovirus, human Boca virus (Human Bocavirus,HBoV), which was also associated with respiratory tract infection in children. At present, the research of HBoV in the world is mainly focused on the establishment of detection methods and epidemiological investigation of HBoV infection. In this paper, the molecular epidemiology of human Boca virus (Human Bocavirus,HBoV) in China was studied, and the molecular biological detection and serological diagnosis of HBoV were preliminarily established in the laboratory. The main goal is to elucidate the prevalence, distribution, variation and correlation with the disease of HBoV virus at the molecular level, and to establish the foundation for the study of serological diagnosis through the expression and purification of antigenic proteins. It can provide reference for further study on the function of viral protein and treatment of virus-related diseases, and provide scientific basis for the possible pathogen detection and monitoring of HBoV. Molecular Detection of HBoV 1) Children hospitalized with Respiratory tract infection The total nucleic acid was extracted from the sample of nasopharyngeal aspiration, The conserved region of the target virus gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). 2) Total nucleic acid was extracted from nasopharyngeal aspiration samples of children hospitalized with respiratory tract infection and reverse transcription. The NS1 gene of human Boca virus was selected as the target gene, the fluorescent quantitative PCR primers and detection probe were designed, and the standard curve was established with the recombinant plasmid as the standard. A fluorescent quantitative PCR method was established for the specific detection of human Boca virus. 2 Epidemiological investigation of HBoV The survey included the occurrence of HBoV in specific populations, Development and disease and health related state and distribution. The results showed that HBoV was mainly associated with bronchitis, pneumonia and bronchopneumonia. The patients were mainly infants under 2 years old. Cloning of HBoV Gene and Analysis and Utilization of capsid variation The cloned HBoV gene and the data in Genbank were used to analyze the variation of HBoV. The expression and purification of HBoV capsid protein (VP1) were analyzed, and the specific region of VP1 (VP1-U) with high antigenicity was selected. The nucleic acid sequence of VP1-U was modified by codon affinity and inserted into pET30a (3 '-terminal His tag). The nucleic acid sequence was transformed into E. coli BL21 (DE3) .IPTG induced expression, ultrasonic lysis of bacteria, nickel column affinity chromatography to obtain the pure product.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
本文编号：2228165
[Abstract]:More than 90% of upper respiratory infections in children and adolescents are caused by the virus. There are many kinds of viruses that cause colds, at least 10 kinds of viruses of more than 200 types in seven viridae. Heikkinen Tu Jarvinen A (2003) thinks that about a quarter of the viruses that cause respiratory tract infections have not yet been found. In recent years, respiratory viruses have frequently erupted or become endemic, the old viruses have mutated and new viruses have been produced. In 2001, Dutch scholars first discovered a new respiratory virus, human metapneumovirus; SRAS outbreak in 2003; and following the outbreak of SARS. Two new coronaviruses were found: human coronavirus (Coronavirus NL63) reported by Dutch scholars and HKU1 (Coronavirus HKU1) first discovered in Hong Kong; In 2005, Swedish scholars reported a new parvovirus, human Boca virus (Human Bocavirus,HBoV), which was also associated with respiratory tract infection in children. At present, the research of HBoV in the world is mainly focused on the establishment of detection methods and epidemiological investigation of HBoV infection. In this paper, the molecular epidemiology of human Boca virus (Human Bocavirus,HBoV) in China was studied, and the molecular biological detection and serological diagnosis of HBoV were preliminarily established in the laboratory. The main goal is to elucidate the prevalence, distribution, variation and correlation with the disease of HBoV virus at the molecular level, and to establish the foundation for the study of serological diagnosis through the expression and purification of antigenic proteins. It can provide reference for further study on the function of viral protein and treatment of virus-related diseases, and provide scientific basis for the possible pathogen detection and monitoring of HBoV. Molecular Detection of HBoV 1) Children hospitalized with Respiratory tract infection The total nucleic acid was extracted from the sample of nasopharyngeal aspiration, The conserved region of the target virus gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). 2) Total nucleic acid was extracted from nasopharyngeal aspiration samples of children hospitalized with respiratory tract infection and reverse transcription. The NS1 gene of human Boca virus was selected as the target gene, the fluorescent quantitative PCR primers and detection probe were designed, and the standard curve was established with the recombinant plasmid as the standard. A fluorescent quantitative PCR method was established for the specific detection of human Boca virus. 2 Epidemiological investigation of HBoV The survey included the occurrence of HBoV in specific populations, Development and disease and health related state and distribution. The results showed that HBoV was mainly associated with bronchitis, pneumonia and bronchopneumonia. The patients were mainly infants under 2 years old. Cloning of HBoV Gene and Analysis and Utilization of capsid variation The cloned HBoV gene and the data in Genbank were used to analyze the variation of HBoV. The expression and purification of HBoV capsid protein (VP1) were analyzed, and the specific region of VP1 (VP1-U) with high antigenicity was selected. The nucleic acid sequence of VP1-U was modified by codon affinity and inserted into pET30a (3 '-terminal His tag). The nucleic acid sequence was transformed into E. coli BL21 (DE3) .IPTG induced expression, ultrasonic lysis of bacteria, nickel column affinity chromatography to obtain the pure product.
【学位授予单位】：贵州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373
【参考文献】
相关期刊论文 前1条
1 程凌鹏;陈森雄;Z.H.Zhou;张景强;;伊蚊浓核病毒三维结构的比较分析[J];中国科学C辑:生命科学;2006年04期
本文编号：2228165
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