鲤鱼外周血白细胞蛋白酶体激活因子PA28全长cDNA的克隆与差异表达分析

Cloning and Identification of Proteasome Activator PA28 Full Length cDNA of Carp (Cyprinus carpio L.)

对DD-RTPCR获得的差异显示片段C15进行地高辛标记后作为探针,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行核酸杂交筛选,从0.8×104个重组噬菌体中,经过2轮筛选获得阳性克隆DM7。测序分析表明,该阳性克隆长1 097 bp,具有完整的开放阅读框架,为编码鲤鱼蛋白酶体激活因子PA28的全长cDNA,编码249个氨基酸,含有PA28的α和β亚单位功能结构域,与已报道的斑马鱼的蛋白酶体激活因子PA28的同源性达93%。分离鲤鱼外周血白细胞,经有丝分裂原在不同条件下刺激后,利用Trizol提取总RNA,根据得到的PA28全长cDNA序列和鲤鱼β-actin的序列,设计引物,采用半定量反转录聚合酶链反应(RT-PCR)方法测定白细胞中PA28基因mRNA表达量的变化。结果表明,白细胞经LPS、ConA刺激后,PA28基因的mRNA表达量有所增加,但并不随时间的延长而持续增加。

The cDNA library of mitogen LPS and ConA stimulated carp（Cyprinus carpio L. ） leucocytes was screened by a DIG labelled DNA probe which contain partial sequence of PA28,this partial sequence was obtained from the DD-RTPCR and consider as the differential express gene of carp leucocytes. After two rounds of screening of 8 000 recombinate phages ,the positive clone obtained has a insert sequence of 1 097 bp in length with an full ORF encoding 249 amino acids of proteasome activator PA28 of carp,including PA28 α and β subunit motifs. The protein sequence showed significant homologues （93 % identity） with zebrafish proteasome activator PA28,which has 248 amino acids. Multiple sequence alignment with other species showed that carp PA28 sequence has the highest homologue with that of zebrafish,and has the lowest homologue with woodchuck, which is 57 % identity. Semi-quantitate RT-PCR indicated the expression of PA28 mRNA in mitogen stimulated carp leucocytes is higher than in normal carp leucocytes,but it did not last as the stimulate time go on,the express of PA28 mRNA in carp leucocytes stimulated by LPS for 4 h is higher than stimulated for 12 h,and the express of PA28 mRNA stimulated by ConA for 4 h is higher than 24 h. The GenBank accession number for the sequence is DQ453126.