Construction And Expression Of Fusional Expressing Plasimd Containing Esat6 Of MTB And GM-CSF Of Human

Objective To clone the Esat6 gene of Mycobacterium tuberculosis H37Rv from pVAE plasmid, thento construct into pIRES with hGM-CSF to form an dicistronic eukaryotic expressing vector pIEG. Totransfect it into Hela cells and then identify it. Our study had laied a foundation for the development of anew type of anti-MTB DNA vaccine which has both preventative and therapeutic effects.Methods 1. Use pVAE as template to amplify Esat6 gene by PCR, then to recombine it with MCS Aof pIRES vector to construct an recombination vector pIEsat6. Use pORF-hGM-CSF vector as templateto amplify hGM-CSF gene by PCR, and to recombine it with MCS B of pIRES to construct aneukaryotic expressing vector pIGM. 2. To check them by Molecule Biology methods. Then, to digest thehGM-CSF gene from pIGM and to recombine it with MCS B of pIEsat6 to construct an eukaryoticexpressing vector pIEG. 3. To transfect pIEG into Hela cells. 4. Identify the expression of Esat6 fromHela transfected with pIEG by SDS-PAGE. 5. Identify the expression of Esat6 from Hela transfectedwith pIEG by IHC methods. 6. Identify the expression of hGM-CSF from Hela transfected with pIEG byELISA methods.Results 1. Accurately cloned the fight size product by PCR and digested the right size DNA segments.The sequencing reports are consistent with the reported. 2. Esat6 protein had been detected bySDS-PAGE. 3. The Esat6 expressing cells had been found by IHC methods. 4. The quantity ofhGM-CSF from the transfected cells was much more than the non-transfected cells'.Conclusion An dicistronic eukaryotic expressing vector containing Esat6 gene and hGM-CSF genewas successfully constructed and then expressed in Hela cells. Our study had laied a foundation for thedevelopment of an anti-MTB DNA vaccine whose preventative and therapeutic effects are better thanBCG.