| The serum of Lampetra japonica contains a large number of immune-related molecules such as C3,C1 q,and VLRB molecules,which participate in natural immunity and adaptive immune defense,and protect the host against a large number of foreign pathogens.The serpin molecule in serum belongs to the complement component and plays an important regulatory role in the complement system by inhibiting the classical activation pathway of complement and the C1 molecule in the lectin pathway.The expression of lamprey serpin(L-serpin)was significantly up-regulated after pathogen stimulation,indicating that L-serpin may be involved in the immune defense process of pathogen infection.The results of sequence analysis indicated that the L-serpin CDS was 1433 bp in length,1371 bp in open reading frame,encoding 456 amino acids,including a serpin domain.L-serpin consists of three ?-sheets and six ?-helices and an exposed reaction center loop(RCL).The amino acid residues of the target protein recognition sites P1 and P1' located in the RCL region are threonine-serine.The bioinformatics software was used to predict and analyze the structural features and physicochemical properties of the L-serpin encoded amino acid sequence.L-serpin is a secreted protein with no transmembrane structure and contains one N-terminal signal peptide and one N-type glycosylation site.Phylogenetic analysis showed that L-serpin belongs to the F branch of the serine protease inhibitor superfamily.The L-serpin gene was amplified by PCR and constructed into p Cold ? prokaryotic expression vector.A large number of r L-serpin proteins were expressed and purified for polyclonal antibody preparation and functional studies.Semi-quantitative analysis and immunoblotting were used to identify L-serpin in the gill,supraneural body,liver,intestine,kidney,heart,leukocytes and seru m of the lamprey,and the expression level in the supraneural body,liver and leukocytes was relatively high.Using real-time quantitative PCR and Western Blot,it was found that the expression of L-serpin was up-regulated in the lamprey after intraperitoneal injection of Vibrio anguillarum,Staphylocccus aureus and Poly I:C,respectively.It is worth noting that the expression of L-serpin was most obvious when stimulated by Vibrio anguillarum,and peaked at 8 h(P<0.01).It was confirmed that L-serpin is mainly localized in the cytoplasm of leukocytes and liver cells by immunofluorescence assays.L-serpin combined with protease recognition active sites P1 and P1' were separately mutated by site mutation method,respectively,followed by protein expression andpurification.The results of surface plasmon resonance and co-immunoprecipitation demonstrated that the L-serpin-WT protein interacts with the lamprey complemental serine protease C1 q protein,while the mutant loses its mutual interactions.By flow cytometry,high content screening test and CCK8 detection experiment,L-serpin can inhibit the killing effect of serum on target cells.In vitro,using QPCR and high content detection methods,overexpression of L-serpin and exogenous addition of r L-serpin in cells inhibited LPS-induced inflammatory factor production and cell death.It was proved that the prokaryotic recombinant L-serpin(r L-serpin)protein can bind to LPS from Gram-negative by immunofluorescence and surface plasmon resonance method.Based on the bacteria binding experiment,L-serpin has the ability to bind to various microorganisms including Gram-negative bacteria and Gram-positive bacteria.In summary,the lamprey serpin belongs to the F-clade of the serine protease inhibitor superfamily,with a typical serpin domain and three-dimensional conformational features.In addition,L-serpin not only participates in the natural immune defense against pathogen infection through the C1 q molecule,but also plays an important role in antibacterial and anti-inflammatory function.Our research provided a new perspective for lamprey immune defense against pathogen infection by complement molecule. |
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