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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,能够诱导较强的抗原特异性Th1型细胞
Objective To construct the bicistronic eukaryotic expression plasmid pIHsp65GM and express it in eukaryotic cells to study the immunogenicity of the gene vaccine and its immunoprotection against Mycobacterium tuberculosis in mice. Methods The gene vaccine was prepared and C57BL / 6 mice were immunized with plasmid DNA in the anterior tibial muscle. After the last immunization, the mice were challenged with the virulent strains of h37rv through the tail vein and the number of colonies of M. tuberculosis in the lung and spleen tissues were counted. The pathological sections of lung and spleen were made. Pathological changes were observed by HE staining. Serum-specific IgG and splenic lymphocyte specific proliferation index (MTT) were measured by MTT method. The level of IFN-γ in the spleen cell culture supernatant was measured, Lactate dehydrogenase (LDH) release assay was used to determine the specific cytotoxic T lymphocyte (CTL) activity of immunized mice. Results The pIHsp65GM gene vaccine was successfully constructed and induced mouse-specific IgG production, spleen lymphocyte proliferation and IFN-γ secretion (mean content was significantly higher than the 2 negative controls (P <0.001)). The average TB load of spleens and lungs in pIHsp65GM-immunized mice was lower than that of the corresponding control organs in the two negative control groups (P <0.001), and significantly higher than that in the BCG immunized control group. Conclusion The plasmid pIHsp65GM was successfully constructed and expressed. This gene vaccine has certain immunoprotective effect against Mycobacterium tuberculosis infection in mice and can induce strong antigen-specific Th1-type cells