鸭疫里默氏杆菌Ⅰ型磷酸二酯酶基因的发现及其分子特征分析  

Discovery and molecular characteristic analysis of the type Ⅰ phosphodiesterase genes from Riemerella anatipestifer

作　　者：陈虹[1] 程安春[2] 汪铭书[2] 朱德康[1] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心,雅安625014 [2]动物疫病与人类健康四川省重点实验室,雅安625014

出　　处：《高技术通讯》2008年第4期436-440,共5页Chinese High Technology Letters

基　　金：国家“十一五”科技支撑计划(2007Z06-017);教育部科技创新工程重大项目培育资金(7060);教育部“新世纪优秀人才支持计划”(NCET-04-0906/NCET-06-0818);四川省基础研究(2006J13-048)资助项目

摘　　要：以兔抗鸭疫里默氏杆菌(RA)阳性血清对 RA 血清1型 CH-1株表达型 DNA 文库中已通过蓝白斑筛选的一个阳性克隆进行了原位杂交筛选。经过5次免疫筛选后,阳性斑经过体内删除,噬菌粒经酶切分析和序列测定,得到一段4434bp 的插入 DNA 片段。运用 NCBI 的 BLASTN、BLASTP、ORF Finder、Conserved Domains、EMBL 的 FASTA 等程序对该片段中的一个命名为 ORF1689的开放阅读框架进行了全面的生物信息学分析。结果显示ORF1689起始密码子上游存在核糖体结合位点,完整 ORF 含1689bp,编码562个氨基酸,其编码蛋白的理论分子量为59.5kD,等电点为5.82。ORF1689具有 phosphodiest 蛋白家族的保守区域,与丙杆菌属的 Caulobacter sp.K31、鞘氨醇单胞菌(Sphingomonas sp.)、Sphin-gopyxis alaskensis 和 Novosphingobium aromaticivorans 的Ⅰ型磷酸二酯酶基因的氨基酸序列同源性分别为49.283%、38.716%、38.693%和38.313%,表明该基因是一种新的Ⅰ型磷酸二酯酶基因。该研究结果对阐明Ⅰ型磷酸二酯酶的生物学功能和 RA 的生理特性与致病机理提供了理论基础和实验数据。A positive clone identified by blue/white selection in the expression DNA library of CH-1 strain of Riemerella anatipestifer （RA） serotype 1 was screened by in situ hybridization using anti-RA rabbit sera. After five rounds of immunoscreening, in vivo excision of a positive phage plague, restriction enzyme digestion analysis and sequencing, an insert sequence of 4434bp was obtained. BLASTN, BLASTP and ORF Finder provided by the American National Center for Biotechnology Information （NCBI） and FASTA provided by the European Molecular Biology Laboratory （EMBL） were applied to the overall bioinfonnatics analysis of one of the sequence＇ s three open reading frames, denominated ORF1689. The result displayed that the complete ORF contains 1689bp and encodes 562 amino acids. The molecular weight is 59.5kD and the theoretical pI is 5.82. A ribosome binding site （RBS） was found in the front of the initiator. The gene contains a conserved domain of phosphodiest. And the amino acid sequence deduced from ORF displayed homologies of 49.283% ,38.716% ,38.693% and 38.313% to those from Caulobacter sp. K31, Sphingomonas sp., Sphingopyxis alaskensis and Novosphingobium aromaticivorans, so the gene is a new Type Ⅰ phosphodiesterase genes. The research result will provide basic theory and experimental data for clarifying the biological function of Type Ⅰ phosphodiesterase genes and the physiological characteristics and pathologic mechanism of RA.

关 键 词：鸭疫里默氏杆菌 Ⅰ型磷酸二酯酶 分子特征分析 

分 类 号：S852.61[农业科学—基础兽医学]