摘要
目的:克隆与肿瘤恶性演进相关但未知功能的新基因MGC39325,构建其真核表达载体,并应用生物信息学初步探讨其功能.方法:应用RT-PCR技术从LoVo细胞中扩增MGC39325基因全长ORF,选用pGEM-T载体进行TA克隆,再将其亚克隆到真核表达载体pcDNA3.1(+)的启动子和终止子之间,通过PCR、限制性酶切分析及测序进行鉴定.应用生物信息学初步分析其染色体定位、蛋白序列、结构域及功能.结果:从LoVo细胞中扩增出1158bp的cDNA,成功进行TA克隆并进一步亚克隆至pcDNA3.1(+)真核表达载体,测序证实为人MGC39325基因.生物信息学分析显示此基因定位于8q12.1,含有2个外显子(1113bp),DNA序列含有表皮样生长因子位点标记(3-14,12-23)、整合素beta链半胱氨酸富集区位点标记(183-196)、铁氧化还原蛋白铁硫结合区位点标记(389-397,1027-1038)、第Ⅷ因子结构域位点标记(105-157,548-591)、羧基端胱氨酸结位点标记(744-782)等,编码由370个氨基酸组成蛋白质,Mr40727.9,pI值为9.45,疏水性平均值为-0.668,含3个低复杂性区域结构,氨基酸序列含有豆蔻酰化位点(19-24,130-135)、cAMP和cGMP4&赖性蛋白激酶(38-41,46-49,204-207)、酪蛋白激酶(49-52,158-161)、酪氨酸激酶(53-61)、蛋白激酶C磷酸化位点(97-99,140-142,208-210)、N-糖基化位点(97-99)等结构域,提示这一新的蛋白质可能在细胞生长、黏附及细胞信号转导方面具有重要功能.结论:成功地克隆了人MGC39325基因全长ORF,构建了其pcDNA3.1(+)真核表达栽体,为进一步研究其功能及其肿瘤中的作用创造了条件.
AIM: To clone human MGC39325, a novel gene possibly involved in tumor progression identified in our laboratory by cDNA microarray analysis of colonic carcinoma and choriocarcinoma, and to construct its recombinant eukary-otic expression vector so as to explore its function and roles in neoplasms. METHODS: Human MGC39325 full length ORF was amplified by RT-PCR from colonic carcinoma LoVo cells and inserted into the vector pGEM-T by TA cloning. Recombinant eukaryotic expression vector (pcDNA3.1-MGC) was then constructed by subcloning technique and confirmed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible structure and function. RESULTS: MGC39325, with 1158bp, was amplified by RT-PCR from human LoVo cells. The eukaryotic expression vector corresponding to MGC39325 (pcDNA3.1 -MGC) was successfully constructed, which was confirmed by RT-PCR and sequencing. Bioinformatic analyses indicated that this gene was mapped on chromosome 8q12.1 with an open reading frame of 1 113 bp. It contained EGF-like domain signature 1 (3-14, 12-23), integrin beta chain cysteine-rich domain signature (183-196), iron-sulfur binding region signature (389-397, 1027-1038), Von Willebrand factor domain signature (105-157,548-591), and C-terminal cys-tine knot signature (744-782) in DNA sequence. It encoded a 370 aa protein as expected. The putative protein, with molecular weight 40 727.9, pl 9.45, and grand average of hydropathicity -0.668, included N-myristoylation site (19-24, 130-135), cAMP- and cGMP-dependent protein kinase phosphorylation site (38-41, 46-49, 204-207), casein kinase II phosphorylation site (49-52,158-161), tyrosine kinase phosphorylation site (53-61), protein kinase C phosphorylation site (97-99, 140-142, 208-210), and N-glycosylation site (97-99). These structural characteristics suggested that it might play a role on cell growth, adhesion and cell signal transduction that may be related to tumor progression and metastasis. CONCLUSION: Human MGC39325 gene has
出处
《世界华人消化杂志》
CAS
北大核心
2005年第9期1059-1064,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No 30170486~~
