结核分枝杆菌ESAT-6 DNA疫苗的细胞和体液应答及保护效率研究  

ESAT-6 DNA vaccine for Mycobacterium tuberculosis and its humoral and T-cell response as well as its protective efficacy

作　　者：蔡宏[1] 田霞[1] 呼西旦[2] 李淑霞[2] 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,新疆乌鲁木齐830000

出　　处：《中国预防兽医学报》2003年第4期279-283,共5页Chinese Journal of Preventive Veterinary Medicine

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 1 3 1 41 )

摘　　要：构建了含分枝杆菌抗原ESAT_6( 6kDa)分泌蛋白 (结构基因 )的真核表达载体pJW43 0 3 ,进行了酶切鉴定和序列分析 ,确定含有正确的读码框。用脂质体介导法将重组表达质粒导入COS7细胞内并瞬时表达 ,收集表达细胞或浓缩上清液 ,进行SDS_PAGE电泳 ,用结核杆菌特异性抗体进行免疫印迹检测 ,证明上述重组质粒能在哺乳动物细胞中表达出特异蛋白。重组表达质粒肌注免疫小鼠 ,首免后 2 1d,未检测到特异性抗体 ,二免和三免 2 1d后ESAT_6的特异性抗体平均效价分别为 1∶40 0和 1∶80 0。三免后 2 1dESAT_6的特异性细胞因子γ_干扰素的浓度为 1 2 .8± 0 .4ng/mL,而阴性对照空载体DNA组三免后只诱导产生 <0 .1ng/mL的γ_干扰素。三免后经静脉强毒攻击 ,免疫组小鼠肺脏的细菌数比对照空载体组减少了 90 %以上。本研究表明 ,该核酸疫苗能同时诱导机体产生细胞和体液免疫应答并获得较高的保护效率。The coding region of M.tuberculosis ESAT_6(6kDa) was cloned into the eukaryotic expression vector pJW4303.The recombinant expression plasmid was confirmed by restriction enzyme digestion and DNA sequencing.COS7 cells were transfected with a mixture containing serially diluted plasmid DNA encoding secreted form of the protein and Lipofectin(Gibco).After incubation,the supernatant and pellet were analysed on 15 % SDS_PAGE gel and the expression of the protein in COS7 cells was demonstrated by westernblot using munoclonal antibody of M.TB H37Rv.Specific antibody titers were determined after C57BL/6 mice were vaccinated by the recombinant eukaryotic expressing vector.The antigen_specific antibody was not detected in sera harvested from immunized mice 21 days after the first injection.Antibody titers of ESAT_6 were 1∶400,1∶800 after second and third infection respectively.After the third injection,Antigen_specific IFN_γ level of ESAT_6 reached to 12.8±0.4 ng/mL.Almost no IFN_γ level of ESAT_6 was detected in control group.After the last immunization with the DNA vaccine,mice were clallenged with 10 6CFU of M.tuberculosis H37Rv via a lateral tail veil,vaccination with ESAT_6 decreased bacterial CFUs in the lung by over 90% compared to control group.The studies indicated that ESAT_6 DNA vaccine could stimulated cell_mediated immune responsed as well as humoral responses.Our data suggested that this DNA vaccine is able to obtain higher protective response against M.TB.

关 键 词：结核分枝杆菌 ESAT-6分泌蛋白抗原 ESAT-6 DNA疫苗 免疫原性 保护 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]