多头带绦虫脑多头蚴cDNA表达文库的构建及初步分析  

Construction and Preliminary Analysis of cDNA Expression Library of Coenurus cerebralis of Taenia multiceps

作　　者：李文卉[1,2] 王建魁 盖文燕[2] 姚菊霞[2] 曲自刚[2] 贾万忠[2] 罗建勋[2] RaduBlaga 付宝权[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点开放实验室,甘肃省动物寄生虫病重点实验室,兰州730046 [3]兰州市七里河区动物疫病预防控制中心,兰州730050 [4]UniversityofAgriculturalSciencesandVeterinaryMedicine,Cluj-Napoca400372,Romania

出　　处：《寄生虫与医学昆虫学报》2011年第3期139-146,共8页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家“十一五”科技支撑计划项目（2007BAD40804）;中央级公益性科研院所基本科研业务费专项（BRF080303）;中国.罗马尼亚政府间科技合作项目（39-18）

摘　　要：从甘肃景泰羊源脑多头蚴原头节提取总RNA，以Oligo（dT）纤维素柱纯化mRNA，利用LambdaZAPIIXR文库构建试剂盒构建了脑多头蚴eDNA表达文库。从构建的原始文库随机挑选单个噬菌斑进行PCR，确定文库重组率和插入外源基因片段大小，鉴定文库质量。结果表明脑多头蚴eDNA表达文库的原始库容量为1．0×10。pfu，扩增后文库滴度为1．6×10^9pfu／mL。随机挑选的165个噬菌斑克隆中，0．25kb以上的克隆155个，重组率为93．9％，对其中0．5kb以上的115个克隆测序共获得104个表达序列标签（EST），分析后得到96个单一EST序列（UniqueEST），其中20个EST与绦虫基因有同源性，38个EST与吸虫基因有同源性，4个EST与线虫基因有同源性，17个EST与其他物种有同源性，其余17个EST没有同源基因。这些EST编码的氨基酸序列有多头带绦虫六钩蚴Tml6抗原、猪带绦虫副肌球蛋白、猪带绦虫免疫原蛋白Ts76等绦虫抗原的同源蛋白以及烯醇化酶等一些酶类、热休克蛋白、肌动蛋白、核糖体蛋白等同源蛋白。Total RNA was extracted from protoscolex of Coenurus cerebralis from naturally infected sheep of Jingtai Co. , Gansu, and mRNA was isolated with Oligo （dT） cellulose column. The Coenurus cerebralis cDNA expression library was constructed with Lambda ZAP II XR vector. The randomly picked clones from primary cDNA library was amplified with PCR to detect the recombinant rate of cDNA library and size of DNA inserts. The results indicated the size of the preliminary cDNA library was 1.0 ×10^6pfu with the titer of the amplified cDNA library of 1.6 ×10^9 pfu/mL. From randomly picked 165 clones, 155 were with the size more than 250 bp and the recombinant rate was 93.9%. Furthermore, 115 clones sized more than 500 bp were sequenced and 104 expressed sequence tags （ESTs） were obtained. After analysis 96 unique ESTs were obtained, of which 20 were homologous with cestode, 38 were homologous with trematode, 4 were homologous with nematode, 17 ESTs showed similarity to known genes of other species, and 17 ESTs had no significant similarity to any sequences available in GenBank databases. Translation products of these ESTs were homologous with T. multiceps oncosphere protein Tml6, T. solium paramyosin, immunogen protein Ts76 and other Taenia antigens, enolase and other enzymes, heat shock protein, actin protein, ribosomal protein and so on.

关 键 词：多头带绦虫 脑多头蚴 CDNA表达文库 表达序列标签 

分 类 号：Q78[生物学—分子生物学]