结核杆菌融合蛋白DNA疫苗构建及免疫保护研究

Construction and Protective Efficacy of the TB DNA Vaccine Expressing Mycobacterium tuberculosis Mtb8.4 and Glutathione S-transferase Fusion Protein

目的:评价结核DNA疫苗免疫鼠产生细胞因子和抵抗结核分枝杆菌攻击的能力。方法:将结核菌Mtb8.4基因和谷胱甘肽S转移酶基因插入pVAX1载体,构建表达Mtb8.4和GST融合蛋白的DNA疫苗pVS8.4G。小鼠分成5组,用pVS8.4G、pVAX1、pIL2S 100μg和PBS 0.1mL各免疫3次,间隔2w。另一组用BCG免疫1次。每组10只鼠在加强后,无菌取脾培养。另外10只小鼠用H37Rv攻击,2w后取脾、肝和肺培养结核菌并计数。结果:pVS8.4G免疫鼠脾细胞培养上清mIL-2和mIFN-γ平均为380.9和422.1pg/mL,显著高于阴性对照组,与BCG组无显著差异。5个组的平均mIL-6和mIL-10无显著差异。pVS8.4G免疫小鼠脾、肝和肺的平均结核菌载量分别为42 093.2、43 264.1和37 264.8CFU/g,低于pVAX1、pIL2S和PBS组相应器官的载量。结论:DNA疫苗pVS8.4G能刺激产生Th1型免疫应答,免疫鼠抵抗H37Rv攻击的能力增强。

Objective： To evaluate the ability to produce cytokines in vitro and the protective efficacy of the mice immunized by the TB DNA vaccine pVS8.4G we constructed. Methods： Plasmid pVS8.4G expressing fusion protein of Mycobaeterium tuberculosis Mtb8.4 and glutathione S- transferase were constructed by inserting mtbS.4 and gst into the vector pVAX1.20 female C57BL/6 inbred mice in 5 groups immunized with the 100ug pVS8.4G, pIL2S, pVAX1, PBS and BCG（105 CFU） for 3 times with 2 weeks interval except BCG（only one injection without boost）. Sera and culture supernatants of spenocytes from 10 mice in each group were detemained for hIL- 2, mIL- 2, mIFG- g, mlL - 6 and mlL- 10 by sandwich ELISA. The other 10 mice in each group were challenged with 106 CFUs of M tuberculosis H37Rv and killed for culturing H37Rv from the spleens, lungs and livers. Results： The concentrations of mIL- 2 and mIFN - γ in the supematants from pVS8.4G immunized mice were 380.9 ± 36.7pg/mL and 422.1 ± 100. 1 pg/mL, significantly higher than tham from the pVAX1, pIL2S, and PBS injected controls （p 〈 0.001 ）. No significantly difference has been observed for the mIL - 6 and rolL - 10 concentrations from the mice in all 5 groups. The average M tuberculosus loads in the spleens, livers and lungs in the pVS8.4G immunized group were 42 093.2 ±3 600.5CFU/g,43 264.1 ± 6 164.2CFU/g and 37 264.8 ± 3 210.5CFU/g respectively. The average bacterial loads in the 3 organs were significantly lower than their counterparts in pVAX1, plL2S and PBS injected groups, but they were significantly higher than their counterparts in the BCG immunized controls. Collusions： TB DNA vaccine pVS8.4G we constructed can evoke Thl immune response which is necessary for prevention against TB. And it is able to enhance protective immunity to H37Rv challenge in the immunized mice.