【作者】 陈明； 李超； 王瑞； 黄婷； 甘西； 余晓丽； 张彬； 雷爱莹； 陈福艳； 梁万文；

【Author】 CHEN Ming1,LI-Chao2,WANG Rui2,HUANG Tin1,GAN Xi1,YU Xiao-li1, ZHANG Bin1,LEI Ai-ying1,CHEN Fu-yag1,LIANG Wan-wen1 1.Guangxi Institute of Fisheries,Nanning 530021 China; 2.College of Animal Science and Technology,Guangxi University,Nanning 530005,China

【机构】 广西水产研究所； 广西大学动物科学技术学院；

【摘要】 为大规模快速克隆罗非鱼细胞免疫功能基因,采用SMART(switching mechanism at 5’end of RNA transcript)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。混合免疫后第3d、5d和7d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,SfiⅠ酶切和CHROMA SPIN-400TM柱分级分离,收集50obp以上的片段与重组于改造的pBluescriptⅡSK载体并转化大肠杆菌,测定文库滴度、重组率及库容量。随机挑取24个克隆进行菌落PCR扩增插入片段。经鉴定,库容量达到1.021×106克隆,原始文库滴度为1078 pfu/ul (1.078×106/ml),重组率94.71%,插入片段大小分布为0.75～3.0kb,平均长度约为1.5kb。随机对24个克隆序列所获得的18条Contigs进行Geneblank Blast分析,得到15条有相关同源信息的Unigene,其中10条为全长cDNA,全长率为66.7%(10/15)。因此,所构建文库的各项指标均达到要求,为筛选罗非鱼免疫功能基因提供了材料来源,且为进一步研究基因的结构和功能奠定了重要基础。更多还原

【Abstract】 In order to fast amply the immune genes of the Tiplia on an 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.Mixed the total RNA of extracted from the 3,5,7 days’ peripheral blood leucocytes after vaccinated,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 amply 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 SfiⅠand size fractionation using CHROMA SPIN-400TM columns,cDNAs(>500bp) were ligated to the SfiⅠdigested,dephosphorylated pBluescriptⅡSK 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 1.02×106 independent clones.The titer of the cDNA library was estimated as 1078 pfu/ul(1.078×106/ml),the rate of recombinant was above 94.71%.The inserts varied from 0.5 to 2.5kb with average size was about 1500bp. 24 clones were sequenced randomly-selected from each library and 18 contigs were acquired.The results of Genebank Blast(n/x) analysis indicated that 15 contigs have the Homologous Gene information which 10 were full-length cDNA and the full-ratio was 66.7%(10/15).The full-length cDNA library meet the requirement of a standard full-length cDNA library.It could lay a strong basis for subsequent work of clone and selection of some immune functional genes.更多还原