我國(guó)人博卡病毒的分子流行病學(xué)及衣殼蛋白獨(dú)有區(qū)表達(dá)純化的研究
發(fā)布時(shí)間：2018-09-07 11:42
【摘要】： 在兒童和少年中，90％以上的上呼吸道感染是由病毒引起的。引起感冒的病毒種類很多，至少涉及7個(gè)病毒科的10多類共200多個(gè)型別的病毒。Heikkinen T，Jarvinen A(2003)認(rèn)為引起呼吸道感染的病毒還有約1／4尚未被發(fā)現(xiàn)。近年來(lái)，呼吸道病毒頻繁爆發(fā)或局部流行，舊的病毒不斷變異，新的病毒不斷產(chǎn)生。2001年，荷蘭學(xué)者首次發(fā)現(xiàn)一種新的呼吸道病毒—人偏肺病毒；2003年SRAS爆發(fā)；繼SARS爆發(fā)后，又有兩種新的冠狀病毒分別被發(fā)現(xiàn)：荷蘭學(xué)者報(bào)道的人冠狀病毒NL63(Coronavirus NL63)及香港首次發(fā)現(xiàn)的人冠狀病毒HKU1(Coronavirus HKU1)；2005年瑞典學(xué)者報(bào)道一種新的細(xì)小病毒—人博卡病毒(Human Bocavirus，HBoV)，同樣與兒童呼吸道感染有關(guān)，已受到廣泛關(guān)注。目前世界各國(guó)對(duì)HBoV的研究主要集中于檢測(cè)方法的建立和HBoV感染的流行病學(xué)調(diào)查。本課題主要研究我國(guó)人博卡病毒(Human Bocavirus，HBoV)的分子流行病學(xué)，在實(shí)驗(yàn)室初步建立HBoV的分子生物學(xué)檢測(cè)方法和血清學(xué)診斷方法。主要目標(biāo)是希望從分子水平來(lái)闡明我國(guó)HBoV病毒的流行、分布、變異及與疾病的相關(guān)性，并通過(guò)抗原蛋白的表達(dá)純化，為建立血清學(xué)診斷方法研究奠定基礎(chǔ)，并可為進(jìn)一步研究病毒蛋白的功能和病毒相關(guān)疾病的治療提供參考，為HBoV可能的病原檢測(cè)監(jiān)測(cè)提供科學(xué)依據(jù)。 一、HBoV分子流行病學(xué) 1、HBoV的分子檢測(cè) 1)從因呼吸道感染而住院的兒童的鼻咽抽吸物樣本中提取總核酸，逆轉(zhuǎn)錄后，PCR擴(kuò)增目標(biāo)病毒基因保守區(qū)，如凝膠電泳顯示該片段并測(cè)序證實(shí)，則可確定為目標(biāo)病毒陽(yáng)性。 2)從因呼吸道感染而住院的兒童的鼻咽抽吸物樣本中提取總核酸，逆轉(zhuǎn)錄。選擇人博卡病毒的NS1基因作為目標(biāo)基因，設(shè)計(jì)熒光定量PCR引物和檢測(cè)探針，以重組質(zhì)粒為標(biāo)準(zhǔn)品建立標(biāo)準(zhǔn)曲線，從而建立特異性檢測(cè)人博卡病毒的熒光定量PCR方法。 2、HBoV流行病學(xué)調(diào)查 調(diào)查包括HBoV在特定人群中的發(fā)生、發(fā)展和疾病及健康有關(guān)狀態(tài)與分布規(guī)律。結(jié)果顯示HBoV主要與支氣管炎，肺炎和支氣管肺炎相關(guān)聯(lián)，，在所檢測(cè)的人群中的發(fā)生率約為8.3％，患者主要為2歲以下的嬰幼兒。 3、HBoV基因的克隆及衣殼變異分析 利用克隆的HBoV基因和Genbank中的數(shù)據(jù)分析HBoV的變異規(guī)律。 二、VP1-U蛋白表達(dá)純化 分析HBoV衣殼蛋白(VP1)序列抗原性，選擇抗原性較高的VP1獨(dú)有區(qū)(VP1-U)表達(dá)。將VP1-U的核酸序列進(jìn)行密碼子親嗜改造，插入到pET30a(+)中(3’末端帶His標(biāo)簽)，轉(zhuǎn)化大腸桿菌BL21(DE3)。IPTG誘導(dǎo)表達(dá)，超聲裂解細(xì)菌，鎳柱親和層析獲得純品。
[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.
【學(xué)位授予單位】：貴州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373
本文編號(hào)：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.
【學(xué)位授予單位】：貴州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 程凌鵬;陳森雄;Z.H.Zhou;張景強(qiáng);;伊蚊濃核病毒三維結(jié)構(gòu)的比較分析[J];中國(guó)科學(xué)C輯:生命科學(xué);2006年04期
本文編號(hào)：2228165
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2228165.html
最近更新
教材專著
