多发性骨髓瘤细胞株ARH-77恶性相关基因hMMTAG2的克隆和序列分析  

Cloning and Sequence Analysis of Tumor-associated Gene hMMTAG2 from Human Multiple Myeloma Cell Line ARH-77

作　　者：田菁燕[1] 胡维新[1] 田尔明 石奕武[1] 申群喜[1] 汤立军[1] 江元山[1] 

机构地区：[1]中南大学湘雅医学院分子生物学研究中心,长沙410078 [2]University of Arkansas for Medical Sciences

出　　处：《生物化学与生物物理学报》2003年第2期143-148,共6页

基　　金：国家自然科学基金资助项目 (No.39880 0 2 1);国家教育部重点实验室访问学者基金资助项目~~

摘　　要：为从人类多发性骨髓瘤细胞株中克隆新的恶性相关基因 ,构建人类多发性骨髓瘤细胞株ARH 77cDNA文库。从ARH 77细胞中提取mRNA并逆转录合成双链cDNA ,经PCR扩增后 ,插入表达载体 ,得到ARH 77细胞cDNA表达文库。将文库菌落印迹至尼龙膜 ,分区培养提取质粒DNA ,建立基因池 ,并分别转染NIH/ 3T3细胞。经G418筛选并计集落数 ,选择克隆数最多的基因池进入第二轮。如此反复进行。通过测序 ,排除所有已知的癌基因、抑瘤基因、细胞因子及其受体 ,获得了有转化活性的cDNA克隆A6 2 17。进一步使用RACE技术对 5′端进行快速扩增 ,获得全长为 130 0bp的cDNA序列 ,该基因由 8个外显子组成 ,编码产物为 2 6 3个氨基酸组成的多肽链。将该基因暂时命名为多发性骨髓瘤转化基因 2 (tumor associatedgene 2fromhumanmultiplemyeloma ,hMMTAG2 )。该基因定位于人类染色体 1q42 .13。生物信息学分析发现 ,hMMTAG2编码有多个蛋白激酶的磷酸化位点、N 肉豆蔻酸化位点及核定位信号 ,提示该基因的表达产物可能是细胞核内的一种信号分子。In order to look for the tumor-associated genes from human multiple myeloma (MM), a cDNA library of human multiple myeloma cell line ARH-77 was constructed with eukaryote expression vector pcDNA3.1(+). The length of inserted fragments in library was 1.2 kb in average. All clones in cDNA library were transferred in situ to nylon membrane, which was divided into eight equal parts (A-H) and cultured in LB medium to set up gene pools. The plasmids in cDNA library and in gene pools were extracted and NIH/3T3 cells were transfected respectively. By G418 screening and colonies counting, gene pool A was chosen for the second cycle transfection. After several cycles, a clone, A62-17, was obtained, which had significant transforming ability. The length of this clone was 993 bp. The RACE technique was used for rapid amplification of A62-17 5′end. The full length of this sequence has 1300 bp and was named as hMMTAG2 gene. hMMTAG2 consists of 8 exons and codes for a polypeptide of 263 amino acids (the accession number in GenBank: AY137773). It was located at chromosome 1q42.13. hMMTAG2 had same transforming activities in NIH/3T3 cells as the clone A62-17, and the number of transformant foci was 6 folds more than the blank vector pcDNA3.1(+). The analysis of bioinformatics revealed that hMMTAG2 had many phosphorylation sites for several protein kinases, N-myristoylation sites and nuclear localization signals, so it may be a signal molecule in the nucleus.

关 键 词：多发性骨髓瘤细胞株 ARH-77 恶性相关基因 hMMTAG2 序列分析 基因克隆 

分 类 号：Q78[生物学—分子生物学]