Molecular Cloning And Tissue And Cell Distribution Of L-CaM In Lampetra Japonica

Calmodulin?CaM?is a kind of important Ca²⁺binding protein which is commonly found in eukaryotes.Calmodulin contains two EF-hand domains binding with Ca²⁺.When Ca²⁺-CaM form a complex,they can bind with the downstream target protein and activate downstream kinase activity,then execute the relevant cell function and biochemical reactions.In this paper,the L-CaM gene with full length sequence of 1592bp was obtained by PCR amplification from the Lampetra japonica.The results of bioinformatics showed that L-CaM had no transmembrane domain and signal peptides,pCold?-L-CaM prokaryotic expression vector was constructed successfully and soluble L-CaM recombinant protein was obtained;the results of mass spectrum confirmed the recombinant L-CaM protein.Circular dichroism?CD?analysis found that?-helix accounted for 89%,no?-fold structure,random coil 11%indicating that The L-CaM secondary structure is mainly composed of?helix.When the Ca²⁺was added to L-CaM protein,the?-helix was reduced to 78%and?-fold was 1%,random coil22%,showing that Ca²⁺can change the composition of L-CaM secondary structure.The construction of pEGFP-N1-CaM eukaryotic expression vector was successfully established,It was confirmed that L-CaM was localized in HeLa nucleus and cytoplasm,LPS and Ca²⁺stimulation affect the transfer of L-CaM from the nucleus to the cytoplasm.These results of Real-time PCR and western blotting showed that mRNA and protein expression of PLA2significantly increased by overexpression of L-CaM in 293T cells,however,the expression of CaMK?was not affected.Immunohistochemistry and immunoblotting assay showed that the L-CaM was highly expressed in intestine?supraneural body,kidney and gill.In conclusion,Ca²⁺-L-CaM complex activates L-CaM,activates the activity of downstream target protein and kinase,affects the process of cell cycle and regulates the immune function.In addition,CaM plays an important regulatory role in cell proliferation,cycle regulation,apoptosis.However,the function of L-CaM from lamprey has not been reported.In this paper,studies of L-CaM molecular structure,tissue localization and overpression research laid a solid foundation for the further research on the function of L-CaM.