Cloning, Expression And Immunological Function Analysis Of L-ClqDC-1from Lampetra Japonicum

The C1q protein family consists of many proteins containing a C1q domain, which existswidely within organisms from bacteria to mammals. The domain organization of theseproteins mainly includes a leading signal peptide, a collagen-like region, and a globular C1qdomain. According to their structural characteristics, the C1q protein family can be dividedinto three subgroups: C1q, C1q-like and ghC1q. They participate in a series of immuneresponses, such as pathogen recognition, phagocytosis, activation of the complement system,and regulation of cell migration depending on versatile gC1q domain which can bind a varietyof self and non-self ligands. C1qDC proteins which are the new important pattern recognitionreceptors have become the potential drugs to regulate the immune response.In the present study, the full-length cDNA (1662bp) of lamprey L-C1qDC-1wasobtained through5’ and3’ end of the rapid amplification experiment, which was composed bythe5’-UTR of107bp,3’-UTR of844bp, and the open reading frame of711bp, encoding236amino acids. We predicted and analyzed physical and chemical properties, structuralcharacteristics and phylogenetic relationships of L-C1qDC-1by bioinformatics methods.The L-C1qDC-1target fragment was subcloned into the prokaryotic expression vectorpColdⅠ, and successfully expressed in RosettaBlue. The soluble L-C1qDC-1recombinantprotein was obtained. Then the recombinant protein was purified by histidine affinitychromatography column. Rabbit against L-C1qDC-1polyclonal antibodies were purified afterthe New Zealand white rabbit was immuned by recombinant protein.Flow cytometry and laser confocal microscopy results showed that the Japanese lampreyimmune body cells could express L-C1qDC-1, and L-C1qDC-1was distributed in thecytoplasm of the cells.L-C1qDC-1could interact with VLRB in lamprey serum by immunoprecipitation andWestern blot analysis. And confocal microscopy detected that when lamprey serum wasadded into cancer cells, L-C1qDC-1and VLRB colocalization phenomenon occured in cancercells, and they positioned in the cell membrane and cytoplasm of cancer cells.In summary, these results indicated L-C1qDC-1might participate in VLRB mediatedimmune responses. They could play an important role in immune responses and defense.