Gene Cloning And Prokaryotic Expression Of HGP44 Functional Domain In Gingipains From Porphyromonas Gingivalis

[Objective]The black-pigmented gram-negative bacterium Porphyromonas gingivalis (P.g) is an important etiological agent of adult periodontal disease.Gingipains are extracellular protease of P.g. They are important virulence factors. HGP44 is adhesin region in the gingipains.it has close relation with the platelet aggregation.The platelet aggregation which is mediated by P.g is dependent on the hgp44 which code the HGP44 protein. The study is on the hgp44 gene clone, recombinant and protein expressions, lay a foundation for the properties of HGP44 and the interaction with the human endothelial cells.[Method]The genomic DNA of P.g was isolated from P.g ATCC 33277. The hgp44 gene fragment was amplified by polymerase chain reaction (PCR),ligated to the cloning vector pMD 18-T and transferred into E.coli DH5a.A postive recombinant plasmid which contained Hgp44 gene had been digested by enzyme Xho I and NdeⅠ,then the hgp44 gene was sub-cloned into expression vector pET 22b and transformed into E. coli BL21(DE3) competent cell. Positive bacterium strain was induced by IPTG, SDS-PAGE reveals that the hgp44 gene was expressed in E. coli BL21(DE3).[Result]The hgp44 gene fragment was amplified by polymerase chain reaction (PCR) was about 1100bp.The recombinant plasmid pET 22b-hgp44 was construced successfully. The SDS—PAGE analysis testified the expression of recombinant protein induced by IPTG is HGP44.[Conclusion]Hgp44 was successfully cloned and expressed in prokaryotic expression system,which lays a foundation for the purification of HGP44 next and the study on its function,furthermore for the interaction of periodontal disease and coronary heart disease.