Expression And Purification Of Recombinant Lampetra Japonica Oral Gland Secreted L251 Protein

To identify the functional gene fragment,a cDNA library was constructed from the oral gland of Lampetra japonica.After random selection of clones for sequencing and BlastX and BlastN programs used for sequence similarity alignment,a neutrophil inhibitory factor(NIF)like protein named L251 was found.L251 belongs to PR-1 subfamily(pathogenesis—related protein),CAP protein superfamily.It contains three structural domain:PR-1domain,a link domain and a cysteine-rich domain.It shares the same functional site with NIF from the Hookworms of adult A.caninum.Sequence specific primers were designed to obtain the full-length cDNA encoding the L251 protein.Then the cDNA was directionally cloned into the pET23b and transformed intoE.coli BL21,and expression was induced by IPTG.After the identification by SDS-PAGE,the recombinant proteins were purified by a specificHis-Tag binding step in which the target protein strongly bind to a chromatography column bearing Ni2+.After denature and renature by dialyzation,the activity of the interaction with neutrophil was tested.L251 was tested to inhibit the migaration of neutrophil through cell culture insert towards a concentration gradient of the chemoattractant fMLP by MIGRATEST CHEMOTAXIS test kit.Meantime,oxidative burst activity was also tested by flow cytometry using dihydrorhodamine(DHR 123)as a fluorogenic substrate.The data suggests that L251 can significantly impaire the mean fluorescence intensity compared to controls.L251 can also prevent the binding ofβ2 integrins on the surface of neutrophil and its FITC-labled monoclonal antibodies.L251 plays an effective role in physiological reaction of neutrophil by as a novel class ofβ2 integrin receptor antagonists.This experiment suggests that L251 protein can protect tnmpetra japonica from host immune response during the invasion,and Lampetra japonica is a novel species which have immune evasion mechanism besides virus and worm etc. This is a new work that clarifies the interactions between the parasites and the hosts in the molecular level,and it provides a promising concept of anti-inflammatory and anti-viral therapy.