Cloning Of Nucleolin-like Gene From Japanese Lampreys And Preliminary Exploration Of Its Role In Innate Immunity

Lampreys are one of the most primitive agnatha vertebrates ever discovered in marine organisms their fossils are found about 360 million years ago,so the lampreys are called "living fossils." Due to their special evolutionary status,the lampreys having great research value for a long time.Nucleolin is an important protein in the nucleolus of eukaryotic cells.It has many important cellular functions and plays a crucial role in cell proliferation,death,immunity,especially in rRNA synthesis and immunity.In this paper,the nucleolin(nucleolin,C23)homolog(nucleolin-like)was discovered in the Janpenese lampetrys(Lampetra japonica)for the first time.The following research results were initially obtained.First,the full-length nucleolin-like gene encoding 646 amino acids was successfully cloned from the leukocyte cDNA library of L.japonica.Through homologous sequence alignment analysis,it was found that this gene had about 50% homology with other vertebrate nucleolins.Meanwhile,we constructed phylogenetic trees of different species of nucleolin,and found that nucleolin-like located between invertebrates and teleosts,which was consistent with the position of lampreys in the evolution of species.Second,the C-terminal fragment of the nucleolin-like was cloned and constructed into the pET-28 a vector to obtain a prokaryotic expression recombinant plasmid.Prokaryotic proteins were expressed and purified in prokaryotic host bacteria.The anti-nucleolin-like polyclonal antibody was prepared by immunizing the New Zealand white rabbits with purified protein,and its titer and specificity were verified.The tissue distribution and expression of nucleolin-like in L.japonica were detected by Real-time PCR,Western Blot and flow cytometry.The results showed that nucleolin-like was expressed in the L.japonica leukocytes and all tissues in mRNA levels,and the expression levels in leukocytes,gill,kidney and supraneural body tissues were more obvious.At last,Poly(I:C)was used to stimulate the leukocytes of L.japonica.The level of nucleolin-like mRNA and protein expression in leukocytes were increased under the stimulation of Poly(I:C).It was also found that the stimulated of poly(I:C)made nucleolin-like transfer from nucleus to cytoplasm.These results suggested that nucleolin-like might play an important role similar to human nucleolin in the innate response of lampreys.In summary,the full-length nucleolin-like gene was first discovered and cloned in L.japonica;anti-nucleolin-like polyclonal antibody was successfully prepared;its distribution in the tissues of L.japonica was ascertained;the expression and translocation of nucleolin-likein cells stimulated by Poly(I:C)were studied.It laid a foundation for further study on the role of nucleolin-like in innate immunity of lampreys.