双顺反子载体构建及其在基因联合免疫中的应用  

Construction of bicistronic vector and its application to combinted DNA vaccine

作　　者：廖国阳[1] 毕胜利[2] 杨建勇[3] 李卫东[1] 陈俊英[1] 张新文[1] 姜述德[1] 

机构地区：[1]中国医学科学院中国协和医科大学医学生物学研究所,昆明650118 [2]中国疾病预防控制中心病毒病预防控制所 [3]卫生部北京生物制品研究所

出　　处：《中华实验和临床病毒学杂志》2006年第2期75-77,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：云南省自然科学基金面上项目(2001C0072M和2004C0070M)

摘　　要：目的为了便于多价DNA疫苗的制备和基因表达的研究。方法以pcDNA3·0为基础构建了pcDNA3·0BA双顺反子载体,将HBVpreS2S与HCVpc154基因插入pcDNA3·0BA构建了单价和双价质粒。质粒转染Cos-7细胞瞬时表达,并经肌肉免疫BALB/c小鼠测定抗体应答和淋巴细胞增殖能力。结果pcDNA3·0BA全长为6·4kb,具有两个外源基因表达单元,其调控元件均为CMV启动子和BGHpolA;两个表达单元中多克隆位点单酶切分别为:MCS1为EcoRⅤ、NotⅠ、XhoⅠ;MCS2为:KpnⅠ、BamHⅠ、EcoRⅠ。双价质粒在Cos-7细胞中preS2S与HCVpc154两个基因都同时表达,诱导小鼠产生了抗HBs和HCV核心蛋白抗体,抗体效价和淋巴细胞对抗原刺激的增殖能力单价和双价质粒相比差异不显著。结论pcDNA3·0BA双顺反子载体可以共表达多个基因,并诱导产生免疫应答。Objective To study preparation of polyvalent DNA vaccine and the control of multiple gene expression.Method A bicistronic vector pcDNA3.0BA was constructed from pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector to form bicistronic expression construct pcDNA3.0BAPC154S2 S and monocistronic expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were transiently expressed in COS-7 ceils and injected into muscles of BALB/c mice. Results pcDNA3.0BA contains two cistronic units, which can co-express two kinds of genes, with the first immunogen gene and the second gene serving as additional immunogen or as modulator for the immune responses. HBV surface Ag and HCV core Ag were coexpressed in vitro. The antibody responses and lymphopriliferation to antigens were similar between bicistronic and monocistronic expression construct in mice. Conclusion pcDNA3. OBA is a novel vecor, which can coexpress two proteins and elicit polyvalent immune responses.

关 键 词：疫苗 DNA 双表达载体 肝炎表面抗原 乙型 肝炎核心抗原 丙型 

分 类 号：R392[医药卫生—免疫学]