摘要
本课题旨在研究结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12(hIL-12)联合免疫小鼠所诱导的细胞免疫应答及对小鼠结核杆菌感染的免疫保护效果.40只C57BL/6N小鼠随机分为Mtb8.4基因疫苗+hIL-12质粒组(联合免疫组)、Mtb8.4基因疫苗组、卡介苗(BCG)组、空载体组和PBS组,基因疫苗、空载体和PBS,经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次.BCG组经尾部皮下注射1×106CFUBCG免疫1次.免疫4周后,每组处死3只小鼠,采用酶联免疫吸附法(ELISA)检测脾细胞培养上清中细胞因子水平;乳酸脱氢酶(LDH)释放法检测细胞毒性T细胞(CTL)杀伤活性.每组其余5只小鼠用结核杆菌H37Rv强毒株经尾静脉攻击,4周后,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果.结果显示,联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平(分别为1493±8pg/ml、748±49pg/ml),显著高于Mtb8.4基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强,对小鼠结核杆菌感染有较好的免疫保护效果,使小鼠肺和脾组织中的结核杆菌菌落数显著减少,组织病变明显减轻,其效果与卡介苗(BCG)组相当,优于Mtb8.4基因疫苗组.表明hIL-12表达质粒与Mtb8.4基因疫苗联合免疫后,能够增强Mtb8.4基因疫苗所诱导的细胞免疫应答,使Mtb8.4基因疫苗的免疫效力得到很大提高.
To study the specific celluar immune response and protective efficacy induced by co-immunization of DNA vaccine of Mtb8.4 and plasmid encoding human interleukin 12 (hIL-12), fourty C57 BL/6N mice were divided into following groups: Mtb8.4 gene vaccine plus plasmid of hIL-12, Mtb8.4 gene vaccine, BCG, vacant vector alone and PBS. Mice were immunized intramuscularly in both hind limbs three times at the intervals of three weeks or once subcutaneously with 1 × 10^6 of viable M. boris BCG Pasteur at the time of the first DNA immunization. Four weeks after the final inoculation, three mice per group were sacrificed to assess cytokine response by ELISA methods and cytotoxic T cell(CTL) activities with LDH release assay and the other five mice per group were challenged intravenously in a lateral tail vein with 1 × 10^6 CFU of virulent M. tuberculosis H37Rv. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile saline. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37℃ until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with the negative control group. The right lung was obtained from each mouse and inflated with and stored in 10% formalin saline immediately. Co-immunization with Mtb8.4 gene vaccine and plasmid encoding hIL-12 induced the secretion of more of Thl cytokines, but not IL-4 and enhanced CTL activity while BCG induced the secretion of both types of cytokines (IFN-7, IL-2 and IL-4). Co-immunization with Mtb8.4 gene vaccine and plasmid encoding hlL-12 could remarkably reduced CFU counts in organs. It is concluded that plasmid encoding human interleukin 12 can improve the specific cellular immune response and protective efficacy induced by the Mtb8.4 gene vaccine alone.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第10期839-844,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
四川省青年科技基金(No.川青科基[2002]1号)~~