Cloning And Function Study Of L-STAT3 In Lampetra Japonica

As the most primitive jawless vertebrate,Lampetra japonica is considered to an important model for studying the origin and evolution of immunity[1].STAT3(Signal transduction and activator of transcription 3)is an important signal transduction and transcription factor,which is involved in many physiological processes such as cell immunity and response.Studies had shown that expression of L-STAT3 was up-regulated after Shewanella sp.immunization,thus L-STAT3 was speculated to be involved in adaptive immune regulation in lamprey.Studies on L-STAT3 of L.japonica have not been reported so far,so the functional study of L-STAT3 can provide a theoretical basis for analyzing the adaptive immune process of L.japonica in response to the immune stimulation of intestinal symbiotic bacteria.Based on this,the research contents in this paper are as follows:1.Identification of differentially expressed proteins in leucocytes of L.japonica in response to Shewanella sp.In this study,label-free quantitative proteomic analysis was used to identify the differentially expressed proteins in leucocytes of L.japonica in response to Shewanella sp..In all,25 differentially expressed proteins were identified.Nine of them were up-regulated and16 were down-regulated.Real-time quantitative PCR(q PCR)was used to verify the expression of the differentially expressed proteins at the transcriptional level.The expression of proteasome subunit protein-5 was consistent in transcriptional and proteomic level of the10 proteins detected.The possible reason for this is that the expression of differentially expressed proteins is regulated at different levels,which leads to the inconsistency at the level of transcription and protein.2.Cloning,bioinformatics and expression pattern analysis of L-STAT3 in L.japonicaAmong all differentially expressed proteins,expression of STAT3 was significantly up-regulated.The full-length of the gene was cloned and sequenced,and the ORF region of the gene was 2124 bp,encoding 708 amino acids.According to the amino acid sequence alignment results,the gene had a high homology with STAT3 from other species,so the gene was named as L-STAT3.The structure and physicochemical characteristics of L-STAT3 amino acids were predicted and analyzed by bioinformatics,and the primary and higher structure of the protein were predicted.A representative species was selected to build theSTAT3 evolutionary tree,and it was found that the L-STAT3 protein and the lamprey are consistent at the evolutionary level.3.Prokaryotic expression of L-STAT3,preparation of polyclonal antibody and expression analysisThe hydrophilic fragments of L-STAT3 were cloned and constructed into p Cold I vector to obtain the prokaryotic expression recombinant plasmid.The protein was expressed and purified in E.coli Rosetta.Anti-stat3 polyclonal antibody was prepared from purified protein and its specificity was verified.At the transcriptional level,L-STAT3 expression was up-regulated in the heart,liver,intestine and muscle,and down-regulated in leucocytes and five other tissues after Shewanella sp.stimulation.The expression of L-STAT3 in L.japonica was detected by Western Blot.The results showed that the expression of L-STAT3 protein in kidney,gill,muscle and leucocytes of L.japonica was relatively high after Shewanella sp.stimulation.The expression of L-STAT3 was up-regulated in leucocytes,gills,supraneural body,and muscles.Immunofluorescence showed that L-STAT3 protein was mainly located in the cytoplasm of supraneural body,and a small amount was found in the nucleus.4.Functional analysis of L-STAT3 in L.japonicaIn order to obtain L-STAT3 active protein,Komagataella phaffii expression system was used to construct and optimize L-STAT3 expression.The L-STAT3 sequence was optimized with rare codons according to K.phaffii gene expression characteristics and constructed into p PICZ?A.The resulting recombinant plasmid was expressed in K.phaffii GS115 and successfully induced by methanol in BMMY.Expression of L-STAT3 recombinant protein reached the highest level in 120 hours under the condition with 1% methanol and 29 o C 250 rpm.In the later stage,the recombinant proteins will be purified and remove the endotoxin,and the effects of different concentrations of recombinant proteins on cells will be explored.In addition,the eukaryotic expression vector of L-STAT3 gene was constructed,and the de-endotoxin recombinant plasmid was instantaneously transfected into HEK-293 T and Hela cells,L-STAT3 overexpression improved the cell survival rate of HEK-293 T and Hela cells.In all,the differentially expressed proteins identified in lamprey leukocytes after Shewanella sp.stimulation were taken as the entry point,which laid a theoretical foundation for the follow-up study on the mechanism of adaptive immune system development in lampreys.The function of L-STAT3 gene was preliminarily investigated,and its effect on promoting the proliferation of HEK-293 t and Hela cells was analyzed,which provide a basis for further exploration its role in lamprey cell proliferation.