采用半巢式PCR技术从临床标本中克隆人类博卡病毒的全基因  

Half nested PCR cloning human bocavirus complete genome from clinical specimen

作　　者：何涛君[1] 王琼[1] 何英[1] 黄烈[1] 张银辉[1] 林广裕[2] 陆学东[1] 

机构地区：[1]广东医学院附属福田医院检验医学部,广东深圳518033 [2]汕头大学医学院第二附属医院儿科,广东汕头515041

出　　处：《国际检验医学杂志》2014年第1期3-5,共3页International Journal of Laboratory Medicine

基　　金：2013年深圳市医疗卫生类科技计划项目(201303158);2013年福田区卫生公益性科研项目(FTWS 201310)

摘　　要：目的为了对广东地区流行的人类博卡病毒生物学特性有更加全面的认识,克隆得到人类博卡病毒全基因,并与国际流行株进行比对。方法采用半巢式PCR分子生物学技术直接从临床呼吸道标本筛查确认人类博卡病毒感染阳性标本中,结合博卡病毒的全基因的生物信息学特点,设计6条特异性引物可配对成5对,通过3轮半巢式PCR方法特异性扩增人类博卡病毒的全基因序列。再将其定向连接到克隆载体中后进行测序,然后向GenBank提交克隆的人类博卡病毒的全基因序列,最后并对人类博卡病毒全基因的生物信息学分析。结果成功克隆并向GenBank提交了3株人类博卡病毒全基因序列,登录号分别为GQ926981、GQ926982和GQ926983。3株人类博卡病毒全基因序列与GenBank里的序列比对,高度同源性达98%以上,均属于人类博卡病毒HBoV1型。结论广东地区流行的人类博卡病毒基因与国际流行株基因比对无大的变异,可为人类博卡病毒相关蛋白的表达工作奠定基础。Objective To clone human bocavirus complete genome,and then compare to international epidemic strains,in order to more recognize bionomics of epidemic human bocavirus in Guangdong area. Methods Clinical specimens were selected screening out human boeavirus infection positive specimens by molecular biology technology, combing with bioinformatics characteristic on human bocavirus complete genome, six specific primers were designed, matched five pairs. After three series half--nested PCR, we can obtain human bocavirus complete gene order. Connecting directional to cloning vector and sequencing,then the complete human bocavirus sequences were submitted to GenBank, and bioinformatics analysis of human bocavirus complete genome was done. Results Three strains complete gene sequences were submitted successfully, and the login numbers were GQ926981, GQ926982 and GQ926983 respectively. Compared with the sequences in GeneBank, the high homology above 98 %, belonging to H BoV type 1. Con- clusion There is no conspicuous gene variation between epidemic human bocavirus in Guangdong area and in international epidemic strains. So it is helpful to express human bocavirus associated proteins in next step.

关 键 词：聚合酶链反应 博卡病毒属 序列分析 

分 类 号：R373[医药卫生—病原生物学]