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将编码结核分枝杆菌MPT-64,Ag85B和ESAT-6分泌蛋白的结构基因或包括信号肽的全基因序列定向克隆到真核表达载体pJW4303中,转化到TOP10大肠杆菌,提取质粒DNA,经限制性核酸内切酶鉴定及序列分析证实含有正确的上述3种分泌蛋白的结构基因（包括信号肽）全序列.用脂质体介导法将重组表达质粒导入COS7细胞内并进行瞬时表达,收集表达细胞或浓缩上清液,12％或15％的 SDS-PAGE电泳分离,用结核杆菌特异性抗体进行免疫印迹杂交,证明上述3种重组质粒能在哺乳动物细胞中表达出特异蛋白.3种重组表达质粒同时肌注免疫小鼠21d后,Ag85B抗体的平均滴度即达到 1:3200,第2次免疫21d后达到1:102400.MPT-64抗体的平均滴度在首免21d后为1:50.第2次免疫21后为1:200.两次免疫21d后均未检测到ESAT-6的特异性抗体.三免21后Ag85B的特异性细胞因子γ-干扰素的浓度为110pg/mL;而 MPT-64和ESAT-6的γ-干扰素浓度未检测到.实验在国内首次用结核杆菌多价DNA疫苗对小鼠进行了免疫实验,二免后的抗体水平已达到或超过国内外已报道的单价苗最高抗体水平,表明多价核酸疫苗和分泌型蛋白载体有利于增强疫苗的保护性应答.
The structural genes encoding the secreted proteins of Mycobacterium tuberculosis MPT-64, Ag85B and ESAT-6 were cloned into the eukaryotic expression vector pJW4303, and transformed into TOP10 Escherichia coli. The plasmid DNA was extracted and restricted Endonuclease identification and sequence analysis confirmed the complete sequence of the structural gene (including signal peptide) containing the above three secreted proteins.The recombinant plasmid was introduced into COS7 cells by liposome-mediated method and transiently expressed The expressed cells or concentrated supernatant were separated by 12% or 15% SDS-PAGE electrophoresis, and immunoblotted with Mycobacterium tuberculosis-specific antibodies to confirm that the three recombinant plasmids could express specific proteins in mammalian cells. The average titer of Ag85B antibody reached 1: 3200 after 21 days of intramuscular immunization with recombinant plasmid, and reached 1: 102400 after 21 days of immunization.The average titer of MPT-64 antibody was 1 after 21 days of first immunization : 50. After the second immunization 21: 1: 200.There was no specific antibody against ESAT-6 detected 21 days after the two immunizations.The specific cytokine interferon-gamma concentration of Ag85B after three-immunization was 110pg / mL; and γ-interferon concentrations of MPT-64 and ESAT-6 Degree was not detected.Experiments in China for the first time with Mycobacterium tuberculosis multivalent DNA vaccine in mice immunization experiments, antibody levels after the two exemptions have reached or exceeded the highest price of monovalent seedlings have been reported at home and abroad, indicating that multivalent nucleic acid vaccine And secreted protein carriers help to enhance the vaccine’s protective response.