title: Study of Hgp44 from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6 and IL-8.
abstract: The aim of this study was to clone, express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis and purify the protein. Furthermore, the effect of Hgp44 from P. gingivalis on inducing HUVECs to secrete IL-6 and IL-8 was evaluated. The Hgp44 gene fragment was amplified by polymerase chain reaction, and then inserted into the cloning vector pMD18-T and linked with a prokaryotic expression vector pET22b to construct the recombinant expression plasmid pET22b-Hgp44. Fusion protein expression was induced by IPTG, and it was purified by immobilized metal-chelating affinity chromatography (IMAC) using an Ni(2+) matrix column. HUVECs were cultured in vitro and different concentrations of Hgp44 were added to confluent HUVEC monolayers and incubated for 2, 8 and 24 h. We extracted the supernatants and then used ELISA kits to test the changes in IL-6 and IL-8 levels. Finally, a 1100-bp fragment was successfully amplified, and the expression of the fusion protein was examined by SDS-PAGE and Western blot analysis, and the data showed that the protein was 44 kDa in size and expressed mostly in the form of inclusion bodies. The purification of the fusion protein was achieved using Ni(2+) affinity chromatography. About 3.5 mg/L fusion protein was obtained. Hgp44 could induce HUVECs to secrete IL-6 and IL-8 levels, which were remarkably increased. In a word, Hgp44 was successfully expressed in a prokaryotic expression system and purified by IMAC using the Ni(2+) matrix column. The effect of Hgp44 in inducing HUVECs to secrete IL-6 and IL-8 was demonstrated.
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: H H
authors.initials: HH
authors.lastname: Cui
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: Q X
authors.initials: QX
authors.lastname: Zhang
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: S L
authors.initials: SL
authors.lastname: Deng
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China.
authors.forename: Y D
authors.initials: YD
authors.lastname: Chen
authors.affiliation: Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, China huic66@hotmail.com.
authors.forename: H
authors.initials: H
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D011993:Recombinant Fusion Proteins