Molecular Cloning,recombinant Expression And Related Immunology Studies Of Mitogen-activated Protein Kinase P38? And ?

P38MAPK is a subclass of the MAPK family and plays a very important role in cell signal transduction in the immune response of higher vertebrates.So far,most of the studies on P38MAPK have been directed at mammals,and it is unclear whether there is P38MAPK and its function in jawless vertebrates.Since lamprey is not only an important representative of the existing jawless vertebrates,it is also an important group of invertebrates that have the significance of initiating the evolution of vertebrates.Therefore,this paper chooses P38MAPK as the research object to explore whether P38MAPK exists in lamprey,and what role it played in its immune defense process.This will not only reveal the molecular evolution of the P38MAPK family from lower to higher vertebrates,but also will lay the foundation for further understanding of the molecular mechanisms of the immune response in jawless vertebrates.In previous transcriptome sequencing experiments,we found that P38MAPK of Lampetra japonica exists in the form of two isoforms P38?(MAPK14)and P38?(MAPK11).We redesigned the primers for MAPK14 and MAPK11 based on the partial sequence of the L.japonica P38?and P38?and their nucleic acid sequences in the genome database of petromyzon marinus,and successfully obtained the open reading frame(ORF region)sequence of L.japonica MAPK14(Lja-MAPK14)and MAPK11(Lja-MAPK11)by the nested PCR method.We used a variety of bioinformatics analysis software to perform various analyses on the primary,secondary,and tertiary structures of their proteins and to construct a phylogenetic tree,and explored the molecular evolution mechanism of the P38MAPK family.These results laid a good theoretical foundation for the design and implementation of the future experiments.On the basis of successful cloning of Lja-MAPK14 and Lja-MAPK11,recombinant expression plasmids of Lja-MAPK14 and Lja-MAPK11were successfully constructed and transformed into expression strain E.coli BL21 for expression.The soluble assays showed that both Lja-MAPK14 and Lja-MAPK11were expressed as inclusion bodies,and both could be detected by immunoblotting using human P38 polyclonal antibody.To further understand the functions of Lja-MAPK14 and Lja-MAPK11,we detected their mRNA and protein expression profiles in response to changes in immune response time by semi-quantitative PCR,real-time quantitative PCR and Western blotting methods.The localization of Lja-MAPK14 and Lja-MAPK11 in lymphocyte-like cells was detected by immunofluorescence.The real-time quantitative PCR and immunoblot results revealed that Lja-MAPK14 and Lja-MAPK11 showed marked upregulation in lymphocyte-like cells and supraneural myeloid bodies after immunization with mixed bacteria.Immunofluorescence assay showed that there was a colocalization between Lja-MAPK14/Lja-MAPK11(P38MAPK)and VLRB~+lymphocytes.At the same time,P38MAPK also appeared in the nuclear after immunization with mixed bacteria.The above experimental results show that Lja-MAPK14 and Lja-MAPK11 are involved in the immune response process of in jawless vertebrates.Our study lays the foundation for further study of the role of P38MAPK in lamprey lymphocytes signal transduction process.