黑素瘤相关抗原D4(MAGE-D4)的生物信息学分析及真核表达载体的构建

Bioinformatics analysis and construction of eukaryotic expression vector of MAGE-D4

目的分析黑素瘤相关抗原D4(MAGE-D4)蛋白的理化性质、结构和功能,构建MAGE-D4真核表达载体。方法通过生物信息学软件分析MAGE-D4蛋白的理化性质、结构和功能。以MAGE-D4/pMAL-C2原核重组质粒为模板,经PCR扩增、酶切后与pEGFP-C1真核表达质粒连接,转化大肠杆菌。连接产物经抗生素筛选、酶切、测序鉴定后,通过脂质体转染A549肺癌细胞。结果MAGE-D4蛋白为不稳定亲水性蛋白,无跨膜结构和信号肽,二级结构以α螺旋为主,具有多个功能性修饰位点,主要定位在细胞核。SLLLVILGV可能为MAGE-D4来源的HLA-A*0201限制性T细胞表位。与MAGE-D4存在潜在相互作用的前3位蛋白依次是染色体成分4非结构维持蛋白同源物A(NSMCE4A)、T细胞识别的黑素瘤抗原1(MLANA/MART-1)、黑素瘤B抗原5(BAGE5)。测序结果证实重组质粒含有MAGE-D4的全长编码序列(CDS),能成功转染A549肺癌细胞。结论MAGE-D4蛋白为不稳定核蛋白,可通过与多种黑素瘤相关蛋白相互作用而发挥功能,其来源的短肽可能具有较强的免疫原性,并构建了MAGE-D4真核表达载体。

Objective To analyze the physicochemical properties,structure and function of melanoma-associated antigen D4(MAGE-D4)protein,and then construct the eukaryotic expression vector of MAGE-D4.Methods The physicochemical properties,structure and function of MAGE-D4 protein were analyzed by bioinformatics.Using MAGE-D4/pMAL-C2 prokaryotic recombinant plasmid as the template,PCR product digested by restriction enzyme was connected with pEGFP-C1 eukaryotic expression plasmid and transformed into E.coli.Ligation products were identified by antibiotic screening,enzyme digestion and sequencing.Then the recombinant plasmid was transfected into A549 lung cancer cells by liposome.Results MAGE-D4 protein was an unstable hydrophilic protein without transmembrane structure and signal peptide.Its secondary structure was mainlyα-helix.MAGE-D4 contained multiple functional modification sites and was mainly located in the nucleus.SLLLVILGV might be a restricted T cell epitope of HLA-A*0201 derived from MAGE-D4.The first three proteins to potentially interact with MAGE-D4 were NSMCE4A,MLANA/MART-1 and BAGE5.DNA sequencing showed that the recombinant plasmid contained full-length coding sequence(CDS)of MAGE-D4 and it could be successfully transfected into A549 lung cancer cells.Conclusion MAGE-D4 protein is an unstable nuclear protein,which may play functions by interacting with a variety of melanoma-related proteins.The peptide derived from MAGE-D4 may have strong immunogenicity.The eukaryotic expression vector of MAGE-D4 has been successfully constructed.