人类博卡病毒HBoV1基因组克隆及启动子活性分析  

Genome cloning of human bocavirus(HBoV1) and analysis of viral promoter activity

作　　者：李京京[1,2] 孙彬[1] 欧阳锦凤[1] 陈莹[1] 韩虎[1] 刘凯于[1] 李毅[1] 

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]湖北师范学院生命科学学院,黄石435002

出　　处：《生物工程学报》2011年第6期909-916,共8页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30670081)资助~~

摘　　要：人类博卡病毒（Human bocavirus,HBoV）是继细小病毒B19之后,第2个被发现可引起人类疾病的细小病毒。通过PCR扩增方法从患有下呼吸道感染的患儿痰液中鉴定HBoV,以鉴定的阳性样本为模板,利用分子生物学方法构建病毒基因组克隆并进行序列分析。2007年10月？2009年3月从湖北省妇幼保健院共收集941例下呼吸道感染患儿的痰液标本,检测到33份HBoV阳性样品,阳性率为3.51%（33/941）;其中1岁以下婴幼儿患者占阳性样72.7%;构建了含有HBoV中间大片段基因克隆WHL-1,基因序列比对为HBoV1型,序列全长5 299 bp（GenBank Acession No.GU139423）;PCR扩增博卡病毒左端唯一启动子区,分别构建病毒启动子增强型绿色荧光蛋白（EGFP）/荧光素酶报告基因重组载体pGL3-pBoV-EGFP/pGL3-Basic-pBoV,转染哺乳动物细胞,通过检测绿色荧光蛋白表达和荧光素酶活性定性并定量研究人类博卡病毒启动子在哺乳动物细胞中的活性;结果显示,启动子在试验细胞中都具有活性,且比强启动子巨细胞病毒启动子（Cytomegalovirus,CMV）活性更高,在293T细胞中HBoV1启动子活性是CMV的4~5倍;已构建的基因组克隆和病毒启动子在哺乳动物细胞中表现的高活性为下一步的病毒转录、翻译分子机制的研究提供了平台。Human bocavirus（HBoV） is a recently discovered parvovirus,which is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in human.In the present study,we screened 941 nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections from October 9,2007 to March 20,2009 in the Children＇s Hospital of Hubei Province.Our results showed that 33 of 941 samples（3.51%） were detected positive for HBoV.To obtain a full-length HBoV clone,three segments which covered the nearly full-length genome were amplified by PCR from HBoV positive samples separately and cloned into pBluescript SKⅡvector,and the resulting plasmid was designated as pWHL-1（GenBank Acession No.GU139423）.We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter,respectively.Our data demonstrated that the HBoV promoter exhibited very high activity in all mammalian cells tested by fluorescent microscopy observation of the EGFP and luciferase activity assay and its strength was 4？5 fold higher compared to that of the CMV promoter.This work provided an excellent tool for further study of the mechanism of transcription and expression of the viral genome.

关 键 词：人类博卡病毒(HBoV1) 克隆 序列分析 启动子活性 

分 类 号：Q78[生物学—分子生物学]