人MAGE-3基因片段的克隆与测序  

Cloning and sequencing of the tumor antigen MAGE-3 gene fragment

作　　者：彭培立[1] 吕颖捷 赵国强[3] 董子明[4] 

机构地区：[1]解放军第155中心医院放射科,河南开封475003 [2]河南省医药学校基础科,河南开封475001 [3]郑州大学基础医学院微生物学与免疫学教研室,河南郑州450052 [4]郑州大学基础医学院病理生理学教研室,河南郑州450052

出　　处：《河南大学学报（医学版）》2005年第2期20-23,共4页Journal of Henan University:Medical Science

摘　　要：目的克隆表达人MAGE-3基因片段(210～623位碱基),以便研究其对MAGE-3阳性恶性肿瘤的生物学作用。方法为选择靶基因,采用分子生物学软件分析MAGE-3基因抗原表位;并设计MAGE-3靶区域引物。从人肺癌组织标本中提取mRNA,用RT-PCR法从中扩增出MAGE-3片段,对其进行XhoⅠⅡ酶切鉴定;将扩增片段克隆于pGEM-TEasy载体中,转化JM109,进行阳性筛选后,运用pGEM-TEasy质粒的T7/SP6作为引物对重组子鉴定,并进行DNA测序。结果抗原表位分析表明MAGE-3基因片段(210～623位碱基)为抗原决定簇丰富区段。RNA体外扩增得到的片段为414bp,并经XhoⅠⅡ酶切鉴定得到证实;pGEM-TEasy-MAGE-3经筛选鉴定正确,DNA测序结果与Genebank比较完全相同,表明靶基因成功插入pGEM-TEasy。结论该片段可用于真核及原核表达。Objective: To clone and express the MAGE-3 gene fragment (210～623nt) for researching its biological effects on MAGE-3 positive malignant tumors. Methods: The antigen epitope of MAGE-3 was analyzed with molecular biology software in order to select the target gene for investigating and the primers of target area in MAGE-3 gene were designed according to the analyzed results. mRNA was extracted from human lung cancer specimen. MAGE-3 gene fragment was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme cutting with XholII did its identification. The PCR fragment was cloned into pGEM-T Easy vector, and then was transformed into JM109. After the selection of positive clones, the primers designed with T7/SP6 sequence of pGEX-T Easy plasmid were applied to identify the recombinant. Further more, DNA sequence analysis of the recombinant was done for the purpose of the correctness of objective fragment cloned. Results: The analysis of MAGE-3 antigen epitopes showed that the MAGE-3 gene fragment (210～623nt) was rich of antigen epitopes. The length of the DNA fragment RT-PCR amplified by RNA was 414 base pairs, which was conformed by enzyme XholII cutting identification. The correctness of linking between MAGE-3 and pGEX-T Easy was verified through positive selection and identification by T7/SP6 primers, and the sequences of DNA fragment were homology with corresponding sequences published in Gene bank, which indicated that the target gene had been inserted into pGEX-T Easy successfully. Conclusion: The MAGE-3 fragment can be used in eukaryotic and prokaryotic gene expression.

关 键 词：基因片段 MAGE-3基因 DNA测序 抗原表位分析 酶切鉴定 生物学作用 分子生物学 癌组织标本 JM109 抗原决定簇 克隆表达 恶性肿瘤 软件分析 mRNA PCR法 扩增片段 体外扩增 原核表达 靶基因 重组子 碱基 阳性 引物 筛选 

分 类 号：R382.31[医药卫生—医学寄生虫学] R734.2[医药卫生—基础医学]