鲤外周血白细胞蛋白酶体激活因子PA28β全长cDNA的克隆、鉴定及其差异表达分析  

Cloning,identification and differential expression analysis of the proteasome activator subunit 2 full-length cDNA from carp (Cyprinus carpio L.)

作　　者：付保忠[1] 卢强[1] 谭业平[1] 孙晓义[1] 李伟[1] 刘相叶[1] 邓洪宽[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,吉林长春130062

出　　处：《水产学报》2008年第6期831-837,共7页Journal of Fisheries of China

基　　金：国家自然科学基金资助项目(30200210;30571433)

摘　　要：采用基因文库筛选方法,克隆了鲤外周血白细胞蛋白酶体激活因子PA28βcDNA,对其序列进行了分析,同时利用半定量反转录聚合酶链式反应(RT-PCR)方法检测了不同外界因子刺激下鲤外周血白细胞PA28β的表达情况。结果显示,用DD-RTPCR的方法获得差异显示片段A18,经地高辛标记作为探针对有丝分裂原刺激的鲤外周血白细胞cDNA文库进行核酸杂交筛选,从0.8×104个重组噬菌体中,经过两轮筛选获得阳性克隆。序列分析表明,该阳性克隆长1175bp,含有一个大小为735bp编码244个氨基酸的完整开放阅读框,为编码鲤鱼蛋白酶体激活因子PA28β的全长cDNA。系统发生分析其与已报道的斑马鱼蛋白酶体激活因子PA28β亲缘关系最近,基因序列的同源性达95%。分离培养鲤外周血白细胞,在不同条件下经PHA和ConA刺激后,利用Trizol提取总RNA,根据得到的PA28β全长cDNA序列和鲤鱼-βactin序列设计引物,利用RT-PCR方法对鲤外周血白细胞PA28β进行差异表达分析,结果表明:经有丝分裂原刺激后前期(4h)白细胞中PA28β的表达量明显增大,但随着时间推移(12h、24h)表达增加量有所降低,并不随时间的延长而持续增加。在刺激的和正常的白细胞中PA28β表达趋势都成抛物线图,不同的是经刺激的比正常的PA28β的表达量提前达到峰值。首次报道鲤蛋白酶体激活因子PA28β的全长cDNA序列,鲤PA28β的cDNA序列GenBank注册号为EU255233,并对其进行差异表达分析,这些结果可为PA28β在鱼类免疫应答中的作用研究提供参考和依据。In order to research the effectiveness of proteasome activator subunit 2 in carp＇s immunologic response, we cloned the cDNA of proteasome activator subunit 2 from peripheral blood leukocyte of carp. Moreover, we also researched the expression information of PA28β in carp＇s peripheral blood leukocyte. The cDNA library of peripheral blood leucocytes which were isolated from carp （Cyprinus carpio L） and stimulated with mitogen PHA and ConA was screened by a probe labelled with DIG. Using DD-RTPCR, the probe considered as the differential expression fragment which is partial sequence of proteasome activator subunit 2 was obtained. After two rounds of screening from 8 thousand recombinante phages, the positive clone was obtained. Sequence analysis indicates that it contains an insert sequence of 1 175 bp in length with full ORF encoding 244 amino acids of proteasome activator subunit 2, including PA28β and partis α subunit motifs. The protein sequence showed significant homologues （95% identity） with zebrafish proteasome activator PA28β, which has 244 amino acids. Multiple sequence alignment with other species showed that carp PA28β sequence has the highest homologue with that of zebrafish, and has the lowest homologue with Xenopus tropicalis, which is 55 % identity. Using the total RNA extracted from peripheral blood leucocyte which was isolated, cultured and stimulated with mitogen PHA and ConA, this paper did semi-quantitate RT-PCR. It displays that the quantity of PA28β in leucocyte stimulated with mitogen PHA and ConA is obviously larger than that in normal carp leucocytes in the prophas（4 h）. However, it is not always much more than that of the normal leucocyte at the same time with the time going, the expression of PA28β mRNA in carp leucocytes stimulated by PHA for 4 h is higher than that stimulated for 12 h, and the expression of PA28β mRNA stimulated by ConA for 4 h is higher than that for 24 h. This is the first report for full length cDNA sequence of the PA28β from carp. The GenBan

关 键 词：鲤 PA28β CDNA克隆 鉴定 差异表达分析 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]