pIHsp65GM的构建及其对结核杆菌感染小鼠的保护

The construction of pIHsp65GM and its protective effects on mice infected by mycobacterium tuberculosis

目的构建pIHsp65GM双顺反子真核表达质粒,并在真核细胞中表达,研究该基因疫苗的免疫原性及其对小鼠感染结核杆菌后的免疫保护效果。方法制备基因疫苗,将C57BL/6小鼠于胫前肌注射质粒DNA免疫,末次免疫后用h37rv强毒株经尾静脉攻击小鼠,计数肺和脾组织中结核杆菌的菌落数,对小鼠的部分肺和脾组织作病理切片,经HE染色观察组织病变的程度,测血清特异性IgG,MTT测定脾淋巴细胞特异性增殖指数,测小鼠脾细胞培养上清中IFN-γ的水平,乳酸脱氢酶(LDH)释放法测定免疫小鼠特异性细胞毒性T细胞(CTL)活性。结果 pIHsp65GM基因疫苗构建成功并且诱导小鼠特异性IgG的产生、脾淋巴细胞增殖以及IFN-γ的分泌(平均含量显著高于2个阴性对照组(P<0.001))。pIHsp65GM免疫组小鼠的脾和肺的平均结核菌载量分别低于两个阴性对照组的相应器官的结核菌载量(P<0.001),同时也显著高于BCG免疫对照组。结论成功构建和表达pIHsp65GM质粒,该基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果。

The purpose of this paper is to study the protective effects of pIHsp65GM plasmid on mice infected by mycobacterium tuberculosis H37 Rv strain and its mechanism.The Hsp65 gene and hGM CSF were respectively amplified by using PCR method;the PCR product was digested by the restriction endonucleases and then ligated to the empty vector pIRES which had digested by the same restriction endonucleases.The recombinant plasmid pIHsp65GM was been expressed,and then used to immunize C57BL/6 mice via intramuscular injection.The mice were sacrificed after the final immunization,and the specific antibody levels were detected by ELISA,and simultaneously spleen lymphocyte proliferation was test.And lungs were harvested for viable mycobacteria quantitation and histopathological examination.Furthermore,following intravenous challenge with virulent M.tuberculosis H 37 Rv,the average concentration of mIFN-γ in the supernatants from pIHsp65GM immunized mice was significantly higher than those from PBS and the vacant plasmid vector-injected controls（P〈0.001）.The average M.tuberculosis loads in the spleens and lungs of the pIHsp65GM immunized group were significantly lower than those of their counterparts in PBS and the vacant plasmid vector-injected groups,but higher than those of their counterparts in the BCG immunized controls.In conclusion,TB DNA vaccine pIHsp65GM can induce Th1 immune response which is necessary for prevention against TB,and it has the ability to enhance protective immunity to H37Rv challenge in the immunized mice.