Construction and identification of the chimeric Mtb8.4/hIL12 eukaryotic expression plasmid

To construct and express the chimeric Mtb8.4/hIL12 eukaryotic expression plasmid, the Mtb8.4-linker was firstly amplified by PCR and then cloned into the single NheⅠand MluⅠcloning sites of pCI-neo, to construct the recombinant plasmid of pCI-neo-Mtb8.4-linker (pML). Later on the hIL-12 gene was amplified by PCR and cloned into the single MluⅠand SalⅠcloning sites of pML and correct pCI-neo-Mtb8.4/hIL12 (pMI) recombinant plasmid was identified by PCR, RE digestion and DNA (sequencing). Finally COS-7 cells were transfected with pcDNA3.1(+)-Mtb8.4 constructs by cationic liposom. Forty-eight hours later, mRNA of targets gene were detected by RT-PCR. The accuracy of pMI plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMI could lead to transient expression of fusion proteins. It concludes that the construction and expression 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.