Cloning Of PKC-?-like Gene From Lampetra Japonica And Study Of Its Protein Function

Lampreys were found as the most primitive jawless vertebrates in marine organisms so far.Due to their particularity of evolution status,lampreys have high research values for a long time.Protein kinase C(PKC)is a kind of phospholipid dependent serine / threonine protein kinase.Protein kinase C-?(PKC-?)is a new member of PKC family,and is widely expressed in animals.PKC-? has been reported to participate in various cellular activities,including cell proliferation,cell death,immunity,and is particularly important in cell apoptosis.There are many researches about PKC-? in human,but few published studies concentrate on lamprey.In this study,we cloned PKC-?-like from lampetra japonica,investigated its functions and got some preliminary results.Firstly,according to the c DNA library of lampetra japonica supraneural body,we successfully cloned the PKC-?-like gene in a totle length of 1770 bp,encoding 589 amino acids.Sequence analysis showed that PKC-?-like was highly homologous with other vertebrates' PKC-?s.By selecting related species,we constructed the phylogenetic trees and found that the lampetra japonica is the most primordial vertebrates,which supports the objective law in biology that animals gradually evolve from the lower to the higher.Secondly,we successfully constructed the recombinant plasmids and get the soluble PKC-?-like recombinant protein by prokaryotic expression and purification.The purified recombinant proteins were used as antigens for immunization of New Zealand white rabbits,to get the rabbit anti lampetra japonica PKC-?-like polyclonal antibody.It was showed that the antibody had good specificity and strong affinity by western blot and flow cytometry.The results of realtime-PCR and western blot indicated that PKC-?-like had the highest expression in lamprey supraneural body.Data from immunofluorescence showed that lampetra japonica PKC-?-like was mainly located in the cytoplasm of supraneural body cells,and a small amount was presented in the nucleus.Moreover,cis-Diaminedichloroplatinum(CDDP),a kind of apopsosis inducers,was employed to stimulate lampetra japonica for the first time.It was found that the levels of the PKC-?-like m RNA and proteins were both increased in supraneural body,suggesting that PKC-?-like may play a crucial role in apoptosis of supraneural body cells.This study provides the basic data for the objective evaluation on the evolution of PKC-?-like in the vertebrates and further study on its functions in apoptosis.