The Study Of Hgp44from Porphyromonas Gingivalis On Inducing HUVECs To Secrete IL-6and IL-8

[Objective]To clone, express the gene of Hgp44in adhesin domains of gingipains from Porphyromonas gingivalis(Pg) and purify the protein. Meanwhile, observing the effect of Hgp44from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6and IL-8.[Methods]The genomic DNA of P.g was isolated from P.g ATCC33277.The Hgp44gene fragment was amplified by polymerase chain reaction (PCR) and then inserted into the cloning vector pMD18-T and sequenced. The correctly fragment was linked with a prokaryotic expression vector pET-22b to construct the recombinant expression plasmid pET22b-Hgp44.The pET22b-Hgp44which had been confirmed by enzyme digestion was transformed into competent E. coli BL21(DE3) cells.Expression of fusion protein was induced by IPTG, and purified by immobilized metal-chelating affinity chromatography (IMAC) using a Ni2+matrix column. SDS-PAGE and Western Blotting analysis were used to examine the fusion protein. HUVECs were cultured in vitro and different concentrations Hgp44were added to confluent HUVECs monolayer and co-cultured for2h,8h and24h. We extracted the supernatants and then used ELISA kits to test the changes of IL-6and IL-8.[Results]A1,100bp fragment was successfully amplified and verified by the agarose gel electrophoresis and sequencing. The generated recombinant expression vectors pET22b-Hgp44were verified by enzyme digestion and agarose gel electrophoresis. The expression of fusion protein in E. coli BL21(DE3) cells was examined by SDS-PAGE and Western Blotting analyses, and the data showed that the protein was44KD in size and expressed mostly in the form of inclusion body. The purification of fusion protein was achieved using Ni2+affinity chromatography. About3.5mg/L fusion protein was obtained.Hgp44could induce HUVECs to secrete IL-6and IL-8levels which were remarkably increased.[Conclusion]Hgp44was successfully expressed in the prokaryotic expression system and purified by IMAC using a Ni2+matrix column.We defined the effect of Hgp44inducing HUVECs to secrete IL-6and IL-8, and lay a foundation for the study of interaction between Hgp44and the human endothelial cells, furthermore for the molecular mechanism of Porphyromonas gingivalis and coronary heart disease.