ABSTRACT
Objective:To construct and identify the chimeric mtb8.4/hIL12 eukaryotic expression plasmid.Methods:Firstly mtb8.4-linker was amplified by PCR and cloned into the single Nhe Ⅰ and Mlu Ⅰ cloning sites of pCI-neo, so pCI-neo-mtb8.4-linker(pML) plasmid was constructed.Secondly hIL-12 was amplified by PCR and cloned into the single Mlu Ⅰ and Sal Ⅰ cloning sites of pML.Finally correct pCI-neo-mtb8.4/hIL12 (pMI) plasmid was identified by PCR,RE digestion and DNA seguencing.Results:The accuracy of pMI plasmid construction was confirmed by a number of molecular biological techniques.Conclusion:The construction of mtb8.4/hIL12 chimera by linkage of M.tuberculosis mtb8.4 gene to human IL12 gene provides the possibility for investigating a new tuberculosis vaccine.
ABSTRACT
Objective To observe the specific cellular immune response induced by co-immunization of DNA vaccine of Mtb8.4 and plasmid encoding human interleukin 12(hIL-12)in mice.Methods Fifteen C57BL/6N mice were divided into following groups:Mtb8.4 gene vaccine plus plasmid of hIL-12,Mtb8.4 gene vaccine,BCG,empty 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?106 of viable M.bovis BCG Pasteur at the time of the first DNA immunization.The level of IFN-? in supernatant of spleno-lymphocyte cultures was measured by ELISA.CTL activities of spleno-lymphocyte were detected with LDH release assay.Results The levels of IFN-? and IL-2 in supernatant of spleno-lymphocyte cultures in the group of Mtb8.4 gene vaccine plus plasmid of hIL-12 were significantly higher than that of group of Mtb8.4 alone(P
ABSTRACT
Objective To prepare Mtb8.4 gene vaccine and to study the cellular immune response induced by the vaccine. Methods The gene encoding Mtb8.4 from Mycobacterium tuberculosis was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1 (+). C57BL/6N mice were vaccinated three times with Mtb8.4 gene vaccine at 3 weeks interval. Four weeks after the final inoculation, mice were sacrificed to assess cytokine response and CTL induction. Results The IFN-? and IL-2 titers were 787.317?45.586pg/ml and 319.953?57.978pg/ml in Mtb8.4 gene vaccine group, 1 486.540?39.600pg/ml and 767.043?50.269pg/ml in BCG group, respectively. The level of IL-4 in BCG group (90.580?10.998 pg/ml) increased significantly as compared to other groups (P