μ-calpain激活因子UK114的克隆与序列分析

Cloning and Sequence Analysis of UK114 of the Activator of μ-calpain

构建了山羊肝脏cDNA文库,采用平板裂解法提取噬菌体DNA,以此为模板用所设计的引物以PCR法扩增出-μcalpain激活蛋白UK114基因,并克隆到pGEM-T easy载体。序列分析表明,UK114 cDNA包括起始密码子和终止密码子在内全长共计1 017 bp,5′非编码区长为39 bp,3′非编码区长为567 bp,编码区长411 bp,编码137个氨基酸序列。与GenBank中Colombo等所得UK114基因比较表明:在5′非编码区,UK114为102 bp,克隆的UK114为39 bp,且二者仅有9个核苷酸序列是相同的;紧接着是起始密码子ATG,然后是一个411 bp的阅读框(40 nt^450 nt),编码137个氨基酸,理论分子量约为15 ku,二者在编码区仅有1个bp不同,为无义突变;在3′非编码区为552 bp,所克隆的UK114为567 bp,二者在此区域有57个核苷酸序列是不同的:UK114为polyA,所克隆的是不同的核苷酸。二者的同源性为91%,突变的86个核苷酸中都为无义突变,开放阅读框中仅有一个核苷酸突变。

Goat liver cDNA library was constructed, and the DNAs of phage were isolated by plate lysate method. The UK114 gene was amplified by polymerase chain reaction , using two primers. and then were cloned into pGEM-T easy vector. Sequencing of the PCR-derived fragments showed that the UK114 cDNA consists of 1 017 bp. The 5＇ non-coding region consists of 39 bp, the 3＇ non-coding region consists of 567bp, and its coding region is about 411 bp （40 nt-450 nt）, which can encode 137 amino acid （14.2ku）. Compared this product with UK114 that Colombo reported, its 5＇ non-coding region consists of 102 bp, and there are 9bp identical. Their encoding region consists of 411 bp, and there is only one mutation nucleotide in coding region, and it is meaningless. The 3＇ non-coding consists of 522bp, the difference between them is that the UK114 of Colombo reported is the poly A tails and the UK114 is the different nucleotides. The homologous was 91%. There are 86 mutation nucleotides, and they are all meaningless. There is only one mutation nucleotide in coding region.