尼罗罗非鱼免疫后外周血白细胞全长cDNA文库的构建及鉴定  

Construction and Identification of Full-length cDNA Library of the Peripheral Blood Leucocytes in Oreochromis niloticus after Vaccination

作　　者：陈明[1] 李超[1] 王瑞[1] 李莉萍[1] 甘西[1] 余晓丽[1] 雷爱莹[1] 黄婷[1] 陈福艳[1] 梁万文[1] 

机构地区：[1]广西水产研究所,广西南宁530021

出　　处：《西南农业学报》2011年第1期329-334,共6页Southwest China Journal of Agricultural Sciences

基　　金：国家科技支撑计划项目(2008BADB9B04);广西科学研究与技术开发计划项目(桂科基0836029);2060302社会公益研究项目(2060302GXIF-2008-03;2060302GXIF-2009-04)

摘　　要：为大规模快速克隆罗非鱼细胞免疫功能基因,采用SMART技术,构建了尼罗罗非鱼链球菌疫苗免疫后外周血白细胞全长cDNA文库。提取免疫后第3、5和7天外周血白细胞总RNA,用PowerscripTM反转录酶逆转录合成第一链cDNA,LD-PCR扩增获得双链cDNA,经SfiI酶切和CHROMASPIN-400TM柱分级分离,收集500 bp以上的片段重组于改造的pBluescript II SK载体并转化大肠杆菌DH5α,测定文库滴度、重组率及库容量。结果表明,构建的cDNA文库原始库容量1.021×106克隆,文库滴度1.078×106pfu/mL,重组率94.71%,插入片段大小0.75~3.0 kb,平均长度约为1.5 kb。随机对24个克隆测序所获得的18条Contigs进行BLASTx分析,发现有15条Contigs有相关同源信息,其中10条为全长cDNA,全长率为66.7%。说明所构建文库的各项指标均达到要求,可为筛选罗非鱼免疫功能相关基因和进一步研究基因的结构和功能奠定基础。In order to rapidly clone the cellular immune function genes of the Tiplia on a large scale,a full-length cDNA library of the peripheral blood leucocytes from the Oreochromis niloticus that vaccinated of S.iniae vaccine by i.p was constructed by using SMART（switching mechanism at 5′ end of RNA transcript） techniques.The total RNA was extracted from peripheral blood leucocytes after vaccinated 3,5,and 7 days.The PowerscriptTM reverse transcriptase was used to synthesize and anchor the first-strand cDNA,and the long distance PCR（LD-PCR） method was used to amplify double-strand cDNA based on the SMART techniques for construction of a full-length cDNA library.The PCR products were digested by proteinase K.After digestion with Sfi I and size fractionation using CHROMA SPIN-400TM columns,cDNAs（500 bp） were ligated to the Sfi I digested,dephosphorylated pBluescript II SK vector.The ligation mixture was transformed into E.coil DH5a.The recombinant vectors were titered and the recombinant rate（blue/white） was determined,and 24 clones were picked randomly from the library for screening size of cDNA inserts through PCR reaction.By identification,the primary constructed cDNA library contained 1.021×106 independent clones.The titer of the cDNA library was estimated as 1.078×106/mL,the recombinant rate was above 94.71 %.The inserts varied from 0.7 to 3.0 kb with average size was about 1500 bp.24 clones were sequenced randomly-selected from each library and 18 contigs were acquired and analyzed by BLASTx.The results indicated that 15 contigs had the homologous gene information which 10 were full-length cDNA with 66.7 % of full-ratio.These results showed that the full-length cDNA library meet the requirement of a standard full-length cDNA library,and it could lay a strong basis for subsequent work of clone and selection of some immune functional genes.

关 键 词：尼罗罗非鱼 外周血白细胞 全长CDNA文库 SMART技术 

分 类 号：S965.125[农业科学—水产养殖]