The Preliminary Research On The Restructuring Lampreys VLRB Protein Function

Lamprey that is always as a representative of the rare jawless vertebrates,has an evolutionary status between invertebrates and vertebrates.Its unique evolutionary status and the special variable VLR variable lymphocyte receptors which mediated adaptive immune system are attentioned by immunologists.There are three distinct VLRs(VLRA ? VLRB ?VLRC).The VLRB is similar to the B lymphocyte from the vertebrates while the others are like the B lymphocyte.Therefore,it is very important to research the VLRs,including the VLRB.The prokaryotic expression vector of p Cold?-VLRB it was constructed in this study and then the corresponding recombinant protein,though inclusion body,with a high purity was obtained.After that,the rabbit polyclonal antibody specific to VLRB of lamprey with high titer was prepared via immunizing the lamprey with the protein,which lay foundation for the successive investigation about its functions.Secondly,the eukaryotic expression vector of p IRES2-Ac GFP1-Nuc-VLRB was constructed and transfected into the mammalian cell line,293 T.The expression of VLRB in the cytoplasm of 293 T was confirmed by the immunofluorescence technique and then that in the secretion by the Western blotting before the soluble VLRB protein was purified from the secretion of the cells transfected for 72 hours by affinity chromatography.It eliminated the misgiving that whether the lacking of post-transcriptional modification in the prokaryotic expression system or insoluble expression may affect the function of VLRB.We analysis the expression and location of VLRB in the tissues of lamprey with the method of Western blotting,immunohistochemistry and immunofluorescence assay.The result shows that VLRB was expressed at relatively higher levels in the kidney,gill,superaneural body though there was VLRB in other tissues including serum,leukocyte,intestines,liver and heart,too.The amount of VLRB expression in the liver,intestines and gill from the lipopolysaccharide-challenging lamprey showed significant difference compared with the non-challenging,which demonstrated that VLRB may play most important roles in the immunologic process of lamprey.No killing effect of VLRB on MCF-7,Escherichia coli and Staphylococcus aureus was found,which suggested that it may not involve itself in the killing effect directly.However,there was interaction of VLRB with C1 q,C3 and intelectin,which implied that VLRB may exert its immunologic function via interacting with these immune-associated molecules.In conclusion,our study shows that VLRB may take part in the immunologic process by interacting with other molecules instead of participating in killing pathogens directly,which lay a solid foundation for the study about the immune system of lamprey later.