带免疫佐剂CEA基因疫苗的构建及其稳定同步表达的研究  

Construction of Carcinoembryonic Antigen (CEA) Gene Vaccine and Stable Coexpression with an Immune Adjuvant

作　　者：黄爱强[1] 赖延东[2] 李秀英[1] 顾锦法[3] 陶莎[1] 蔡卫斌[1] 谢金卫[1] 罗超权[1] 

机构地区：[1]中山大学中山医学院生物化学教研室,广东广州510089 [2]广州医学院化学致癌研究所,广东广州510050 [3]中国科学院上海生物化学研究所,上海200031

出　　处：《癌症》2004年第3期282-287,共6页Chinese Journal of Cancer

基　　金：广东省科技计划项目(No.2002C30305);广西科技攻关项目(No.0235024-23)~~

摘　　要：背景与目的:基因疫苗是目前肿瘤免疫基因治疗研究的热点。目前癌胚抗原阳性肿瘤的治疗效果不佳,本研究拟利用质粒pVAX1构建带有免疫佐剂白细胞介素2同步表达的癌胚抗原(carcinoembryonicantigen,CEA)基因疫苗,探讨一种新的肿瘤生物治疗方法。方法:利用RT-PCR从患者肿瘤组织中钓取CEA目的基因cDNA;运用分子克隆技术,通过内部核糖体进入位点(IRES),将CEAcDNA和hIL-2cDNA连接于质粒pVAX1中,采用化学发光免疫法和ELISA双抗体夹心法分别检测CEA抗原和hIL-2因子的表达;同时用蛋白质分析软件分析和预测目的抗原的特性。结果:测序结果证实本实验所构建的重组质粒插入DNA序列无错配,质粒上所接入的CEAcDNA与GenBank公布的M29540和M17303的序列99.8%同源,经蛋白质分析软件预测,其抗原性、跨膜结构片段、信号肽位点和二、三级结构特征与同源癌胚抗原基本一致。hIL-2cDNA与母本序列100%同源。检测结果说明该重组质粒在体外CHO细胞株中可同步表达CEA和hIL-2分子。结论:从肿瘤组织中成功地获取了CEAcDNA,所构建的pVCEA2重组质粒能在体外细胞中同步表达CEA和IL-2蛋白分子,这为进一步体内实验研究基因治疗CEA阳性肿瘤提供了新的可能途径。BACKGROUND & OBJECTIVE:Carcinoembryonic antigen (CEA) positive c ancers are poorly responded to different kinds of treatments. Gene vaccines are promising in research of gene immunotherapy for these tumors.In this study, a CE A gene vaccine with hIL-2 as an immune adjuvant was constructed into a pVAX1 ve ctor for synchronous expression, so as to explore experimentally a new biotherap y strategy against tumors. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), CEA cDNA was obtained from a large intestine carcinoma tiss ue; its encoded protein was compared with the CEA presented in GenBank using the protein analysis software. The acquired CEA cDNA fragment was linked to hIL-2 cDNA via an IRES site and cloned into the pVAX1 vector. The recombinant plasmid was estimated by CEA luminometry assay and hIL-2 ELISA measurement respectively . RESULTS:The nucleotide sequences of the target gene fragments of the recombina nt plasmid were verified. The acquired CEA sequence is highly homologous with M2 9540 and M17303 (99.8%) in GenBank; and the PCR sequence of hIL-2 is coinciden t with the original cDNA (100%) provided. The antigenicity,membrane-spanning s egments, signal cleavage sites, secondary structure and 3D structure of the acqu ired CEA protein were similar to the original proteins of M29540 and M17303 pred icted by the protein analysis software. Results showed the recombinant could ste adily express CEA antigen and hIL-2 protein synchronously in CHO cells in vitro . CONCLUSION: The CEA cDNA was obtained from the tumor tissue and the CEA gene v accine with hIL-2 coexpression was constructed successfully. It has provided a possible method for immunotherapy against CEA positive cancers in vivo.

关 键 词：免疫佐剂 CEA基因 疫苗 基因表达 治疗 

分 类 号：R730.3[医药卫生—肿瘤]