鲤鱼白细胞介素-8全长cDNA的克隆、鉴定及其差异表达分析  

Cloning,identification and differential expression analysis of the interleukin-8 full-Length cDNA from carp

作　　者：谭业平[1] 孙晓义[1] 卢强[1] 李莲瑞[2] 邓洪宽[1] 付宝权[3] 

机构地区：[1]吉林大学人兽共患病教育部重点实验室,吉林长春130062 [2]新疆塔里木大学动物科技学院,新疆阿拉尔843300 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《中国兽医学报》2008年第6期667-671,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30200210,305714330)

摘　　要：用DD-RTPCR的方法获得差异显示片段A26,经地高辛标记作为探针对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库核酸杂交筛选,从0.8×104个重组噬菌体中,经过2轮筛选获得阳性克隆。序列分析表明该克隆含有1个大小为294bp编码98个氨基酸的完整开放阅读框,氨基酸序列含有Chemokine-CXC功能结构域,前体中含有22个氨基酸组成的引导肽,氨基酸序列比对显示成熟白细胞介素-8(IL-8)多肽中第12、13和14构成CXC基序,但之前无ELR基序。系统发生分析其与荷兰鲤鱼亲缘关系最近,氨基酸序列的同源性达89%。分离培养鲤鱼外周血白细胞,在不同条件下经LPS、PHA和ConA刺激后,提取总RNA,根据鲤鱼IL-8全长cDNA序列和β-ac-tin序列设计引物,利用半定量反转录聚合酶链式反应(RT-PCR)方法对鲤鱼外周血白细胞IL-8进行差异表达分析,结果显示,经有丝分裂原刺激后前期(4h)白细胞中IL-8的表达量明显增大,但随着时间推移(12、24h)并不一直比同期正常白细胞表达量大,表达量趋势成峰形图。The cDNA library of peripheral blood leucocytes which were separated from carp(Cyprinus carpio L.)and stimulated with mitogen was screened by a probe labelled with DIG.Using DD-RTPCR the probe considered as the differential expression fragment which is partial sequence of interleukin-8 was obtained.After two rounds of screening from 8 thousand recombinate phages,the positive clone was obtained.Sequence analysis indicates that it contains a insert sequence of 609 bp in length with an ORF encoding 98 amino ac...

关 键 词：鲤鱼 IL-8 CDNA克隆 鉴定 差异表达分析 

分 类 号：Q78[生物学—分子生物学]