Construction and identification of the chimeric mtb8.4/hIL12 eukaryotic expression plasmid

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.