结核分枝杆菌DNA三价苗的细胞与体液应答研究  

The triplevalent DNA vaccine for Mycobacterium tuberculosis and its immunogenicity

作　　者：蔡宏[1] 田霞[1] 李淑霞[2] 胡勤[1] 朱玉贤[1] 

机构地区：[1]北京大学生命科学学院生化与分子生物学系,北京100871 [2]新疆畜牧科学院兽医研究所

出　　处：《中国人兽共患病杂志》2003年第5期22-26,共5页Chinese Journal of Zoonoses

基　　金：国家高技术研究发展计划 ( 863计划 )资助项目 (批准号 :2 0 0 1AA2 13 14 1)

摘　　要：本文构建了含分枝杆菌抗原Ag85B(30kDa) ,MPT - 83(2 6kDa)和ESAT - 6 (6kDa)分泌蛋白的真核表达载体pJW 4 30 3,酶切鉴定和序列分析 ,确定含有正确的读码框。重组表达质粒导入COS7细胞内并瞬时表达 ,证明上述三种重组质粒能在COS7细胞中表达出特异蛋白。三种重组表达质粒同时肌注免疫小鼠 ,首免 ,二免和三免小鼠后 2 1dAg85B抗体平均滴度分别为 1∶32 0 0 ,1∶5 12 0 0和 1∶10 2 4 0 0 ,MPT - 83抗体平均滴度二次免疫和三次免疫后 2 1d测得为 1∶2 5和 1∶5 0 ,三次免疫后均未检测到ESAT - 6特异性抗体。在三免后 2 1dAg85B和MPT - 83的特异性细胞因子γ -干扰素的浓度分别 2 75 pg/ml±16 5 ,2 2 0 pg/ml± 19 8;而ESAT - 6的γ -干扰素浓度为 2 2 0 pg/ml± 9 9。本研究表明 ,多价核酸疫苗能同时诱导机体产生细胞免疫应答和体液免疫应答 ,可能有利于增强疫苗对抗结核病的抗性。The coding regions of M.tuberculosis Ag85B(30kDa),MPT-83(26kDa)and ESAT-6(6kDa)were cloned into the recombinant eukaryotic expression vector pJW4303.The recombinants were confirmed by enzyme digestion and DNA sequencing.COS7 cells were transfected with DNA encoding secreted forms of these three proteins,the expression of these 3 proteins in COS7 cells was demonstrated by immunoblot using monoclonal antiserum of M.TB H37Rv.Specific antibody titers were determined 21days after C57BL/6 mice were vaccinated by the three recombinant eukaryotic expressing vectors.Ag85B induced autibody titers were 1∶3200,1∶51200 and 1∶102400 after the first,second and third injection respectively.Very low antibody titer(1∶25,1∶50)specific for MPT83 was produced after the second and third injection,and we recorded no antigen-specific antibody titer against ESAT-6 in sera harvested from immunized mice 21days after the third injection.Antigen- specific IFN-γlevel of Ag85B,MPT-83 and ESAT-6 were 275±16.5 pg/ml and 220±19.8 pg/ml and 220±9.9 pg/ml 21days after the third injection.In conclusion,The triplevalent DNA vaccines had stimulated cell-mediated immune responses as well as humoral responses.Our data suggests that polyvalent DNA vaccines are able to enhance protective response against M.TB.

关 键 词：结核分枝杆菌 三价DNA疫苗 细胞 体液 免疫应答 AG85B MPT-83 ESAT-6 

分 类 号：R378.911[医药卫生—病原生物学] R392[医药卫生—基础医学]