Molecular Evolution Of L-MASP1-like And Its Role In Mediating Immune Regulation Of Lamprey

The complement immune system of vertebrates is a plasma proteolytic cascade system,which is the body's first line of immune defense against pathogens invading the outside world.The system involves the interaction between more than 30 different group groups,among which the enzyme as the core member is a serine protease containing a multifunctional domain.The complement system mainly includes three activation pathways: classic,bypass,and lectin.The mannose-binding lectin pathway is an important pathway that is activated at the initial stage of infection.Before an immune response occurs in an organism without a specific immune response,the lectin complement pathway plays a major defensive role.Mannose-binding lectin-related serine protease-1(MASP-1)is the central protease molecule in the lectin pathway.Current scientific research results show that the main components of mannose-binding lectin-related serine proteases include MASP-1,MASP-2 and MASP-3 molecules.This thesis takes the lamprey MASP-1 molecule,a model organism derived from adaptive immune evolution and development studies,as the research object,and systematically analyzes the molecular evolution between this molecule and MASP-1 and MASP-2 derived from other species.The difference,explored the role of lamprey MASP-1 molecules in the regulation of immune response and mediating cell apoptosis,and provided a theoretical basis for studying the mechanism of lamprey MASP-1 molecules in the process of immune defense.The main conclusions of the paper are as follows:1.Molecular cloning and molecular evolution of lamprey MASP-1In this paper,based on the MASP homologous sequences retrieved from the self-built lamprey library in the laboratory,the positive and negative primers were synthesized by the biological company,and the six tissues of the lamprey,including the heart,liver,intestine,kidney,gill,and pith,were synthesized.c DNA was used as a template for PCR reaction,and the molecule was successfully cloned in lamprey.Sequence analysis showed that the ORF region encoded by this gene contains 723 amino acids,encoding more than 20 amino acids.Using SMART,Prot Param and other bioinformatics software and online website system to predict and analyze the structural composition,physical and chemical characteristics of the amino acid encoded by the molecule,and the structure of the protein.Next,we used this molecule to compare homologous sequences between different species with MASP family members MASP-1 and MASP-2,which showed that the molecule has more than 80% identity with MASP family members MASP-1 and MASP-2.Source and homologous segments are the main functional domains.Next,the biology software MEGA7.0 was used to establish a phylogenetic tree.The results of the phylogenetic tree showed that this molecule is divided into a branch with the MASP family molecule MASP-1.Therefore,it is reasonable to speculate that this molecule may be more similar to MASP-1.It is speculated that its function may be related to the functions of MASP-1 and MASP-2 and is closer to the functions of MASP-1.In order to further verify this speculation,motif analysis,exon analysis,and collinearity analysis were performed on the molecular sequence,and the results all showed that the molecule is closer to MASP-1 derived from other species.Based on the above analysis,the molecule was named L-MASP1-like.2.Subcellular localization of L-MASP1-like and its role in immune response regulationIn order to explore the expression of L-MASP1-like in various tissues of lamprey,the expression of L-MASP1-like was expressed in various tissues in lamprey,and was mainly distributed in liver,intestine,and kidney as reveaed by Quantitative Real-time PCR(q PCR)and western-blot.After lamprey was stimulated by LPS,Shewanella and Aeromonas,the expression of L-MASP1-like was up-regulated in tissues such as liver,intestine and kidney compared with the control group(PBS).Stimulation with Staphylococcus aureus and Vibrio anguillarum can also regulate expression of L-MASP1-like in liver,and significant up-regulation of L-MASP1-like could be detected in Staphylococcus aureus immune-stimulation group.The above results all show that L-MASP1-like participates in the immune response process of lampreys,and the expression of L-MASP1-like molecules in the liver of lampreys is higher.Next,the lamprey liver tissue was digested and fixed,and the lamprey liver tissue and marrow tissue cells were separated.The subcellular localization analysis of L-MASP1-like was performed by immunofluorescence technology.The confocal results showed that the localization results of L-MASP1-like in the two tissue cells were consistent,widely distributed in the cell.3.The role of L-MASP1-like in mediating cell apoptosisL-MASP1-like in lamprey serum was neutralized using MASP-1 polyclonal antibody,and the effect of lamprey serum on MCF-7 cells before and after neutralization was analyzed.Flow cytometry analysis showed that serum containing L-MASP1-like molecules induced apoptosis of MCF-7 cells,while serum that neutralized L-MASP1-like molecules did not show the effect of promoting apoptosis.Therefore,it is speculated that L-MASP1-like molecules mediate and promote the apoptosis of MCF-7 cells.In order to further identify the apoptotic effect of L-MASP1-like molecules on MCF-7 cells,the key proteins Bax,Bcl-2,Caspase3 and Caspase8 related to the apoptotic pathway were selected,and three in MCF-7 cells treated with different serum were tested.The expression of this apoptosis-related protein further verified that L-MASP1-like can promote the apoptosis of MCF-7 cells.In summary,this study systematically analyzed the molecular evolution of L-MASP1-like,which provided a theoretical basis for the study of the evolution of lampreys serum complement system.The functional analysis of L-MASP1-like showed that this molecule is involved in the regulation of the immune response of lampreys and mediates the apoptosis process of MCF-7 cells,which provided a reference for further exploration of the mechanism of L-MASP1-like molecules involved in lamprey lectin pathway.