FI Proteins Immune Activity Preliminary Study From Lampetra Morii

Lampetra morii is the most primitive jawless vertebrate and is an important model organism for studying the origin and evolution of immunity.The FI molecule(L-FI)in the serum is a complement component and plays an important role in inhibiting the C3 molecule in the classic complement activation pathway,lectin pathway and bypass activation pathway.The open reading frame(1893bp)of lamprey FI(L-FI)gene encodes a polypeptide of 618 amino acids,and L-FI contains KAZAL(kazal-type protease inhibitor module domain),SR(scavenger receptor cysteine-rich domain),LDLr1,LDLr2(two class a low-density lipoprotein receptor domain)and Tryp?SPc(trypsin-like serine protease domain)domains.In this paper,the open reading frame(ORF)of L-FI was obtained by PCR amplification and constructed into the prokaryotic expression vector p Cold I.The L-FI protein was successfully expressed in E.coli and a polyclonal antibody against lamprey FI was prepared.The antibody titer and specificity were determined by enzyme-linked immunosorbent assay(ELISA)and western blotting,respectively.Using semi-quantitative,western blotting and immunofluorescence methods,L-FI was mainly distribute in the liver,intestine and leukocytes of lamprey,and localized in the nucleus and cytoplasm of liver cells and leukocytes.The results of mass spectrometry and co-immunoprecipitation showed that L-FI interacted with L-C3 protein.The experiments of native L-FI and L-C3 depleted from the lamprey serum were performed,depleted of L-FI and L-C3 serum groups inhibited killing of target cells.The results of flow cytometry,confocal laser scanning and cell deposition demonstrated that L-FI affected the deposition of L-C3 on the cell surface.According to the results of q PCR,both L-FI and L-C3 showed a trend of up-regulation after bacterial stimulation.Our results laid the foundation for the study of the lamprey complement molecular,and provided important data for the origin and evolution of the lamprey immune molecular.