人博卡病毒HBoV结构蛋白基因的克隆、原核表达、纯化及VP1在杆状病毒中的表达
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摘要
下呼吸道感染能引起儿童高发病率,是导致儿童死亡的重要病因之一。在2005年10月瑞典科学家Allander等从下呼吸道感染婴幼儿的分泌物中发现了一种新的细小病毒,它是人支气管炎的一种病原体,该病毒能引起儿童上、下呼吸道感染,甚至可引起严重的呼吸道疾病。从分子生物学、系统发育树分析和流行病学等多方面综合研究表明,该病毒具有细小病毒亚科的基本特征,并与细小病毒亚科的博卡病毒属成员结构特征相似,从而将其归类为博卡病毒属的新成员,并命名为人博卡病毒。
人博卡病毒是一种无包膜的、单链DNA病毒,基因组DNA长度大约5299 nt,它是继细小病毒B19之后发现的第二种对人类致病的细小病毒,到目前为止已有18个国家和地区的研究者证实了人博卡病毒感染在儿童呼吸道疾病中普遍存在,部分研究者还在血清、粪便以及尿液的标本中也检测到了人博卡病毒,推测该病毒与胃肠道疾病有关。人博卡病毒可以单独感染,也可以与其他病毒混合感染,感染可全年发生,但深秋、冬季和早春为感染多发季节。目前对人博卡病毒生物学特性及其在人类疾病中的意义仍不清楚。
本文根据我们实验室克隆得到的大片段人博卡病毒序列设计VP1和VP2引物(该序列已经提交GeneBank,登录号为：GU 139423),通过PCR扩增得到结构蛋白基因。并将VP1和VP2基因分别都克隆到原核表达载体pMAL-c2X和pET28a,转化大肠杆菌DH5α和BL21(DE3),经IPTG诱导表达融合蛋白,通过Westen Blot检测目的蛋白的表达。利用Amylose亲和层析柱和Ni柱纯化目的蛋白,并用纯化后得到的目的蛋白免疫新西兰大白兔,制备多克隆抗体,从而为进一步研究该病毒结构蛋白基因的转录和翻译机制提供可靠的工具。
此外,为了弄清楚结构蛋白VP1独特区(VP1-u)是否可以单独表达,还是从整个结构蛋白VP1表达后被裂解下来的。本实验将人博卡病毒主要衣壳蛋白基因VP1克隆到杆状病毒表达转移载体(pFastBacHTA),通过转座获得重组Bacmid DNA,用这种DNA转染昆虫细胞Sf9,转染7到10天细胞完全破裂后,扩大培养使重组病毒达到一个比较高的滴度,收获重组病毒粒子,分别应用His-tag抗体和兔抗HBoVVP1-U高价免疫血清进行Western Blot,以检测VP1蛋白除了自身表达外,它所包含的VP1-U蛋白是否也进行了表达。结果表明VP1独特区不能单独表达。
Lower respiratoty tract infection (LRTI) is the most common disease of children, and it is a leading cause for the mortality of children. In October of 2005, a new parvovirus was first detected by Allander from nasopharyngeal aspirates of children with lower respiratoty infection, it is a pathogen of bronchiolitis and also can induce upper or lower respiratoty infection of children, even severe respiratoty disease. Studies from its molecular biology, analysis of phylogenetic tree and epidemiology indicated that this new virus has basic characteristics of the family Parvoviridae and shares the similar structural feature with the members of the other known bocavirus genus of subfamily Parvovirinae, suggesting that this virus belongs to a new member of the Genus Bocavirus, named Human Bocavirus (HBoV). Nowadays, the genus Bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and the recently identified human bocavirus (HBoV).
HBoV is a non-enveloped single-stranded DNA virus, the current genomic DNA reference sequence of HBoV is 5299 nt in length and it is the second one of parvoviruses which cause human disease after human parvovirus B19. Studies from all over world showed that HBoV infection generally existed in children with respiratory tract illnesses and were also detected in serum, fecal and urine samples, indicating that the virus was related with gastrointestinal tract disease. HBoV alone can infect people and also co-infect people with other viruses. HBoV infection occurs through the whole year, but it usually occurs in the cold season, such as late autumn, winter and early spring. At present, the biological features of HBoV and its significance of human disease by this virus remain unknown.
In this study, the forward and reverse primers were designed based on the reported structural protein gene VP1and VP2 of HBoV genome isolated by our laboratory (Accession number of GeneBank:GU 139423). These two genes were amplified by using PCR and inserted into prokaryotic expression vector pMAL-c2x and pET28a and then transformed into the Escherichia coli DH5a strain and BL21(DE3), respectively. The fusion protein expression was induced by IPTG and identified by Western Blot with MBP-tag antibody and His-tag antibody. The target fusion protein was then purified by Amylose affinity chromatography and Talon Resin Column. The purified VP2 protein was used to immunize the New Zealand rabbit to prepare an antibody against this protein.
To find out whether the VP1 unique part of the VP1 protein is expressed alone or cleaved from the VP1 protein, the full length capsid protein VPlgene was inserted into the baculovirus expression transfer vector (pFastBacHTA) to obtain the recombinant Bacmid DNA which was then transfected into insect cell Sf9.7-10 days post transfection, the supernatant of transfected-cells was used to infect Sf9 cells to get more viruses with higher titer. The recombinant VP1 protein was identified by Western Blot with His-tag antibody or hyper-immune serum against VP1-U of HBoV. Our results showed that no VP1 unique part of VP1 protein was produced from the recombinant baculovirus.
人博卡病毒是一种无包膜的、单链DNA病毒,基因组DNA长度大约5299 nt,它是继细小病毒B19之后发现的第二种对人类致病的细小病毒,到目前为止已有18个国家和地区的研究者证实了人博卡病毒感染在儿童呼吸道疾病中普遍存在,部分研究者还在血清、粪便以及尿液的标本中也检测到了人博卡病毒,推测该病毒与胃肠道疾病有关。人博卡病毒可以单独感染,也可以与其他病毒混合感染,感染可全年发生,但深秋、冬季和早春为感染多发季节。目前对人博卡病毒生物学特性及其在人类疾病中的意义仍不清楚。
本文根据我们实验室克隆得到的大片段人博卡病毒序列设计VP1和VP2引物(该序列已经提交GeneBank,登录号为：GU 139423),通过PCR扩增得到结构蛋白基因。并将VP1和VP2基因分别都克隆到原核表达载体pMAL-c2X和pET28a,转化大肠杆菌DH5α和BL21(DE3),经IPTG诱导表达融合蛋白,通过Westen Blot检测目的蛋白的表达。利用Amylose亲和层析柱和Ni柱纯化目的蛋白,并用纯化后得到的目的蛋白免疫新西兰大白兔,制备多克隆抗体,从而为进一步研究该病毒结构蛋白基因的转录和翻译机制提供可靠的工具。
此外,为了弄清楚结构蛋白VP1独特区(VP1-u)是否可以单独表达,还是从整个结构蛋白VP1表达后被裂解下来的。本实验将人博卡病毒主要衣壳蛋白基因VP1克隆到杆状病毒表达转移载体(pFastBacHTA),通过转座获得重组Bacmid DNA,用这种DNA转染昆虫细胞Sf9,转染7到10天细胞完全破裂后,扩大培养使重组病毒达到一个比较高的滴度,收获重组病毒粒子,分别应用His-tag抗体和兔抗HBoVVP1-U高价免疫血清进行Western Blot,以检测VP1蛋白除了自身表达外,它所包含的VP1-U蛋白是否也进行了表达。结果表明VP1独特区不能单独表达。
Lower respiratoty tract infection (LRTI) is the most common disease of children, and it is a leading cause for the mortality of children. In October of 2005, a new parvovirus was first detected by Allander from nasopharyngeal aspirates of children with lower respiratoty infection, it is a pathogen of bronchiolitis and also can induce upper or lower respiratoty infection of children, even severe respiratoty disease. Studies from its molecular biology, analysis of phylogenetic tree and epidemiology indicated that this new virus has basic characteristics of the family Parvoviridae and shares the similar structural feature with the members of the other known bocavirus genus of subfamily Parvovirinae, suggesting that this virus belongs to a new member of the Genus Bocavirus, named Human Bocavirus (HBoV). Nowadays, the genus Bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and the recently identified human bocavirus (HBoV).
HBoV is a non-enveloped single-stranded DNA virus, the current genomic DNA reference sequence of HBoV is 5299 nt in length and it is the second one of parvoviruses which cause human disease after human parvovirus B19. Studies from all over world showed that HBoV infection generally existed in children with respiratory tract illnesses and were also detected in serum, fecal and urine samples, indicating that the virus was related with gastrointestinal tract disease. HBoV alone can infect people and also co-infect people with other viruses. HBoV infection occurs through the whole year, but it usually occurs in the cold season, such as late autumn, winter and early spring. At present, the biological features of HBoV and its significance of human disease by this virus remain unknown.
In this study, the forward and reverse primers were designed based on the reported structural protein gene VP1and VP2 of HBoV genome isolated by our laboratory (Accession number of GeneBank:GU 139423). These two genes were amplified by using PCR and inserted into prokaryotic expression vector pMAL-c2x and pET28a and then transformed into the Escherichia coli DH5a strain and BL21(DE3), respectively. The fusion protein expression was induced by IPTG and identified by Western Blot with MBP-tag antibody and His-tag antibody. The target fusion protein was then purified by Amylose affinity chromatography and Talon Resin Column. The purified VP2 protein was used to immunize the New Zealand rabbit to prepare an antibody against this protein.
To find out whether the VP1 unique part of the VP1 protein is expressed alone or cleaved from the VP1 protein, the full length capsid protein VPlgene was inserted into the baculovirus expression transfer vector (pFastBacHTA) to obtain the recombinant Bacmid DNA which was then transfected into insect cell Sf9.7-10 days post transfection, the supernatant of transfected-cells was used to infect Sf9 cells to get more viruses with higher titer. The recombinant VP1 protein was identified by Western Blot with His-tag antibody or hyper-immune serum against VP1-U of HBoV. Our results showed that no VP1 unique part of VP1 protein was produced from the recombinant baculovirus.
引文
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[2]周仁彬林创兴林广裕人博卡病毒的研究进展国际呼吸杂志2008年第28卷第18期
[3]Tattelsatl, P., Bergoin,M.,Bloom,M.E., et al. Family Parvoviridae//Fauquet CM, Mayo MA, ManiloffJ, eds.Virus taxonomy:classification and nomenclature of viruses Eighth report of the International Committee on the Taxonomy of Viruses.New York:Elsevi er Academie Press.2005:353-369
[4]李晶哲人细小病毒-博卡病毒的研究进展国际病毒学杂志2008年6月第15卷第3期
[5]何慧(综述),新发现的呼吸道感染病原——人博卡病毒医学综述2008年11月第14卷第21期
[6]Kapoor, A., Slikas, E., Simmonds, P., Chieochansin, T., Naeem, A., Shaukat, S.,et al. A newly identified bocavirus species in human stool.J Infect Dis2009; 199:196-200.
[7]Arthur, J.L., Higgins,G.D., Davidson, G.P., Givney, R.C., Ratcliff, R.M. A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog 2009; 5(4):e1000391.
[8]Kapoor, A., Slikas, E., Simmonds, P., et al. A newly identified bocavirus species in human stool. J Infect Dis 2009,-199:196-200.
[9]AmitKapoor,Peter., Simmonds,Elizabeth. Slikas,Linlin, Li., Human Bocaviruses Are Highly Diverse, Dispersed,Recombination Prone, and Prevalent in Enteric Infections. The Journal of Infectious Diseases 2010; 201(11):1633-1643
[10]郑丽舒段招军人博卡病毒——呼吸道感染病新病原 传染病信息2007年第20卷第2期
[11]罗迪贤,刘巧突,林应标,等.人类博卡病毒基因组序列与进化分析[J].实用预防医学,2006,13(6)：1430-1432
[12]Thaweesak, Chieochansin., Salin,Chutinimitkul., Sunchai Payungporn, Thitikul Hiranras, Rujipat Samransamruajkit, Apiradee Theamboolers, Yong Poovorawan Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV) Virus Research 129 (2007) 54-57
[13]漆正宇,瞿小旺,刘文培,等.人博卡病毒基因克隆及衣壳编码基因序列变异分析[J].病毒学报,2007(11),23(6)：447-152
[14]Natalie, Brieu., Bernard, Gay., Michel,Segondy., and Vincent, Foulongne.* Electron Microscopy Observation of Human Bocavirus (HBoV) in Nasopharyngeal Samples from HBoV-Infected Children JOURNAL OF CLINICAL MICROBIOLOGY,Oct.2007,p.3419-3420
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[17]Bastien, N.,Brant,K., Dust, K.,et al. Human Bocavirus infection. Canada. Emerg Infect Dis,2006,12(5):848-850
[18]Smuts, H.,Hardie,D. Human Bocavirus in hospitalized children, South Africa. Emerg Infect Dis,2006,12(9):1457—1458
[19]Arden,K.E.,McErlean,P.,Nissen,M.D.,et al Frequent detection of human rhinoviruses,paramyxovirus,coronavirus,and bocavirus during acute respiratory tract infections. [J]. Med virol,2006,78(9):1232-1240
[20]Lin F, Zeng A, Yang N, et al. Quantification of human bocavirus in lower respiratory tract infections in China. Infect Agents Cancer,2007,2:3
[21]Quxw, DuanZJ, QizY, el al. Human bocavirus infection, People's Republic of China. Emerg Infect Dis,2007,13(1):165-168
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[2]周仁彬林创兴林广裕人博卡病毒的研究进展国际呼吸杂志2008年第28卷第18期
[3]Tattelsatl, P., Bergoin,M.,Bloom,M.E., et al. Family Parvoviridae//Fauquet CM, Mayo MA, ManiloffJ, eds.Virus taxonomy:classification and nomenclature of viruses Eighth report of the International Committee on the Taxonomy of Viruses.New York:Elsevi er Academie Press.2005:353-369
[4]李晶哲人细小病毒-博卡病毒的研究进展国际病毒学杂志2008年6月第15卷第3期
[5]何慧(综述),新发现的呼吸道感染病原——人博卡病毒医学综述2008年11月第14卷第21期
[6]Kapoor, A., Slikas, E., Simmonds, P., Chieochansin, T., Naeem, A., Shaukat, S.,et al. A newly identified bocavirus species in human stool.J Infect Dis2009; 199:196-200.
[7]Arthur, J.L., Higgins,G.D., Davidson, G.P., Givney, R.C., Ratcliff, R.M. A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog 2009; 5(4):e1000391.
[8]Kapoor, A., Slikas, E., Simmonds, P., et al. A newly identified bocavirus species in human stool. J Infect Dis 2009,-199:196-200.
[9]AmitKapoor,Peter., Simmonds,Elizabeth. Slikas,Linlin, Li., Human Bocaviruses Are Highly Diverse, Dispersed,Recombination Prone, and Prevalent in Enteric Infections. The Journal of Infectious Diseases 2010; 201(11):1633-1643
[10]郑丽舒段招军人博卡病毒——呼吸道感染病新病原 传染病信息2007年第20卷第2期
[11]罗迪贤,刘巧突,林应标,等.人类博卡病毒基因组序列与进化分析[J].实用预防医学,2006,13(6)：1430-1432
[12]Thaweesak, Chieochansin., Salin,Chutinimitkul., Sunchai Payungporn, Thitikul Hiranras, Rujipat Samransamruajkit, Apiradee Theamboolers, Yong Poovorawan Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV) Virus Research 129 (2007) 54-57
[13]漆正宇,瞿小旺,刘文培,等.人博卡病毒基因克隆及衣壳编码基因序列变异分析[J].病毒学报,2007(11),23(6)：447-152
[14]Natalie, Brieu., Bernard, Gay., Michel,Segondy., and Vincent, Foulongne.* Electron Microscopy Observation of Human Bocavirus (HBoV) in Nasopharyngeal Samples from HBoV-Infected Children JOURNAL OF CLINICAL MICROBIOLOGY,Oct.2007,p.3419-3420
[15]Choi,E.H., Lee, H.J., Kim, s.l., et al. The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children,2000-2005. Clin Infect Dis,2006,43(5):585—592
[16]Allander,T.,Jartti, T.,Gupta,S.,et al.Human bocavirus and acute wheezing in children. Clin Infect Dis,2007,44(7):904-910
[17]Bastien, N.,Brant,K., Dust, K.,et al. Human Bocavirus infection. Canada. Emerg Infect Dis,2006,12(5):848-850
[18]Smuts, H.,Hardie,D. Human Bocavirus in hospitalized children, South Africa. Emerg Infect Dis,2006,12(9):1457—1458
[19]Arden,K.E.,McErlean,P.,Nissen,M.D.,et al Frequent detection of human rhinoviruses,paramyxovirus,coronavirus,and bocavirus during acute respiratory tract infections. [J]. Med virol,2006,78(9):1232-1240
[20]Lin F, Zeng A, Yang N, et al. Quantification of human bocavirus in lower respiratory tract infections in China. Infect Agents Cancer,2007,2:3
[21]Quxw, DuanZJ, QizY, el al. Human bocavirus infection, People's Republic of China. Emerg Infect Dis,2007,13(1):165-168
[22]赵林清,钱渊,朱汝南,等.北京地区婴幼儿急性呼吸道感染与新近报道的人细小病毒相关的初步研究.中华微生物学和免疫学杂志2006,26(5)：385-388
[23]Lau,S.K., Yip, C.C.,Que, T.L., et al. Clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples children in Hong Kong.J Infect Dis.2007,196(7):986-993
[24]Kupfer, B., Vehreschild, J., Comely, O., et al.Severe pneumonia and human bocavirus in adult[J] Emerg Infect Dis,2006,12(10):1614-1616
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