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

The cDNA library of Lamprey was established by our laboratory in early, a neutrophil inhibitory factor(NIF)-like protein, called L251, which isolated from the buccal glands of Lampetra japonica. L251 is belong to Cysteine-rich secretory protein(CRISPs), Pathogenesis-related protein 1 (PR-1) by structure prediction and sequence analysis. CRISPs are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. The complete conserved sequence and functional sites between L251 and NIF were determined by protein and cDNA analysis.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 specific His-Tag binding step. L251 was tested to inhibit the migaration of neutrophil through cell monolayer in Transwell towards a concentration gradient of the chemoattractant fMLP. Meantime, neutrophils was separated by Neutrophil Separating Medium of Human and Lymphocyte Separating Medium of Human. The pEFGP-L251 vector was constructed for transfection. We transfected the pEGFP-L251 into HUVECs by Liposome 2000 and tested by Fluorescence Microscope, and constructed a cell monolayer model by Transwell. The results which detected by Flow Cytometry showed that L251 shared the same activity with NIF.It has long been known that parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation. Parasite have evolved different strategies to subvert the host defense mechanisms in favor of their own well-being. To begin and sustain the infection process ,it is essential that these parasites avoid the host's early reactions to injury that is often mediated by circulating leukocytes and chemoattractants derived from activated endothelial cells or the complement system. L251 should be useful in the design of therapeutically effective anti-inflammatory agents, and suggest novel therapies to treat hookworm infections.