A Preliminary Study On The Gene Cloning And Protein Function Of Lamprey CDK-2-like

Lamprey is the most important representative of vertebrates.From the point of view of genetic information,its genome is relatively primitive,which plays a vital role in studying the origin and evolution of vertebrates.The most ideal model creature for vertebrates.CDK-2 is a Ser / Thr protein kinase,a member of the cyclin-dependent protein kinase(CDKs)family,which is expressed in various tissues of the animal body and has multiple biological functions.Cell cycle regulation,DNA replication,DNA damage,apoptosis and aging play an important role and are closely related to the occurrence of various diseases.CDK-2 has been extensively studied in other jawbone vertebrates,but little is known about its potential role in jawless vertebrates.This paper cloned the full-length sequence of lamprey CDK-2-like for the first time,and preliminary explored the protein function,and achieved the following research results: First,this paper successfully cloned a length of 897 bp in the c DNA library of lamprey white blood cells,CDK-2-like full-length gene encoding 298 amino acid residues.The homologous sequence alignment analysis of CDK-2-like found that CDK-2-like has about 80% homology with CDK-2 of other jawed vertebrates.The construction of CDK-2 phylogenetic trees of different species indicates that lamprey CDK-2-like is located between vertebrates and invertebrates,which is in line with the objective law of gradual evolution from lower animals to higher animals.Second,the paper successfully constructed a prokaryotic expression plasmid for CDK-2-like.After prokaryotic expression and protein purification,soluble pure CDK-2-like recombinant protein was obtained.New Zealand white rabbits were immunized with pure recombinant protein as antigens to produce highly specific CDK-2-like polyclonal antibodies.Western blot and flow cytometry results showed that the antibody has good specificity and strong affinity.Real-time fluorescence quantitative PCR(Realtime-PCR)and Western blot results showed that CDK-2-like was expressed in gonadal gonadal tissues with high levels.Through immunofluorescence and laser confocal experiments,it was found that CDK-2-like is mainly located in the cytoplasm and nucleus of lamprey white blood cells.Again,the paper explores the role of CDK-2-like in cell apoptosis and finds that Act-D-induced apoptosis can reduce CDK-2-like transcription and protein expression levels.Overexpression of CDK-2-like can inhibit Act-D-induced apoptosis of HEK-293 T cells.Adding the CDK-2 inhibitor PHA-848125 to normal lamprey whiteblood cells can induce apoptosis,and can slightly reduce the expression of CDK-2-like.In addition,we also found that Act-D and CDK-2 inhibitor PHA-848125 have a synergistic effect in inducing apoptosis,and PHA-848125 can help Act-D reduce the expression of CDK-2-like to a certain extent CDK-2-like is closely related to apoptosis.The above studies provide an experimental basis for objectively evaluating the evolution of CDK-2-like in vertebrates and proving its role in apoptosis.