تنسيل مورثة الصدمة الحرارية GroEL لدى اليرسينيا و التعبير البروتيني لها باستخدام بلاسميد التعبير البروتيني pHEN6

الملخص EN

Yersinia enterocolitica is a gram negative, pathogenic, facultative intracellular bacterium.

It parasites on macrophages and grows in phagosomes, in which a group of proteins plays critical roles in tolerating stressful conditions within the cell.

One of these important proteins is the heat shock protein (HSP) GroEL.

In this work, we aimed to produce a recombinant GroEL protein by gene cloning and protein expression and purification, in order to use the recombinant protein in the future to study its function in the microbe life cycle and the possibility to use it against the yersinosis.

Furthermore, beside being vital for macrophage penetration during infection, GroEL is one of the most immunodominant proteins of Yersinia.

Genomic DNA from Y.

enterocolitica was extracted from a PCR-confirmed strain using Yersinia Multiplex PCR.

From this genomic DNA, the GroEL gene was amplified by PCR using specific primers that contain the restriction sites XbaI and BstEII at 3’ and 5’ ends, respectively.

The amplified and digested GroEL DNA fragment, as well as the digested pHEN6 plasmid, were ligated and then used to transform E.

coli competent cells.

The positive colonies expressed a soluble rGroEL protein (60 kDa) in the periplasmic space and this recombinant protein could be purified by metal affinity chromatography, as demonotrated by electrophoresis (SDS-PAGE 10%) and gel staining or immunoblotting using 6x-His specific antibody.