葡萄球菌肠毒素A和黑色素瘤抗原A3基因重组真核共表达载体的构建及其在293T细胞的表达  

Construction of eukaryotic coexpression vector of recombinant MAGE-A3originating from laryngocarcinoma and Staphylococcal endotoxin A with stable expression of vector in 293T cells

作　　者：吉晓滨[1] 吕景礼[2] 陈敬贤[3] 刘启才[2] 谢景华[1] 

机构地区：[1]广州市第一人民医院耳鼻咽喉科,广东广州510180 [2]广州医学院实验医学研究中心,广东广州510182 [3]美国哥伦比亚大学医学院病理与细胞生物系,美国纽约10032

出　　处：《中国耳鼻咽喉头颈外科》2012年第7期349-353,共5页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：广州市科技局资助项目(2009JI-C501-2)

摘　　要：目的构建葡萄球菌肠毒素A(staphylococcal endotoxinA,SEA)和喉癌来源的黑色素瘤抗原A3(melanoma-associated antigenA3,MAGE-A3)基因共表达的真核表达载体,检测、鉴定其在293T细胞中的表达情况。方法分别用RT-PCR方法 及人工化学合成法获得MAGE-A3和SEA基因片段,然后依次将MAGE-A3和SEA基因克隆至含内部核糖体进入位点的真核表达载体IRES序列的上、下游,构建成重组质粒pMGEA3-IRES-SEA。经脂质体转染至293T细胞以后,用荧光定量PCR、蛋白免疫印迹分别鉴定SEA和MAGE-A3基因的表达。结果限制性内切酶酶切分析证实MAGE-A3和SEA基因均正确地克隆在pMAGEA3-IRES-SEA中,基因测序结果与GenBank公布的MAGE-A3、SEA序列完全一致,实现了双基因的正确重组。重组质粒转染293T细胞后能检测到MAGE-A3、SEA基因的高水平的有效表达。稳定表达双基因的293T细胞上清对外周血单个核细胞(peripheral blood mononuclear cell,PBMC)增殖有促进作用,与细胞中SEA的表达和分泌有关。结论成功地构建了pMAGEA3-IRES-SEA真核表达质粒,并能在293T细胞中有效表达MAGE-A3和SEA蛋白,由此奠定了其作为抗喉癌DNA疫苗应用的基础。OBJECTIVE To construct an eukaryotic coexpression vector of Staphylococcal endotoxin A（SEA）and human melanoma-associated antigen gene A3（MAGE-A3）originating from laryngocarcinoma by using genetic recombinant method,and to identify its expression in 293T line cells.METHODS MAGE-A3 transcripts were linked into pMD18-T vector by RTPCR.SEA gene fragments were artificially synthetized by chemical way.MAGE-A3 gene and SEA gene were subcloned in turn into ribosome in eukaryotic expression vector,then were linked by IRES（Internal Ribosome entry site,IRES）between upstream and downstream of the site sequence.pMAGEA3-IRESSEA recombinant plasmid was constructed,then was transfected into 293T line cells.Their gene expressions were identified by fluorescent quantitative RT-PCR and protein immunoblotting respectively.RESULTS The sequence of MAGE-A3 and SEA carried by pMAGEA3IRES-SEA were perfectly identical with that reported in GenBank.It was confirmed that MAGE-A3 and SEA gene were correctly cloned into vector by restriction enzyme digestion and nucleotide sequencing.Correct reconstructuring of p-MGEA3-IRES-SEA was achieved.The effective expression with high level of MAGE-A3 and SEA were demonstrated by immunoblot after they were transfected into 293T cells.Supernatant of 293T cells with stable expression of these two genes had promoting effect on proliferation of peripheral blood mononuclear cell（PBMC）,which had relations with expression and secretion of SEA in 293T cells.CONCLUSION The eukaryotic expression plasmid pMAGEA3-IRES-SEA wasconstructed successfully and was capable of effective expression of MAGE-A3 and SEA protein in 293T line cells.This work provided a basis for the application of pMAGEA3-IRES-SEA as a DNA vaccine candidate inlaryngocarcinoma immunotherapy.

关 键 词：葡萄球菌属 超抗原 真核细胞 基因 喉肿瘤 克隆 分子 

分 类 号：R739.65[医药卫生—肿瘤]