Research Of The Interaction Of Microcystin With Cyanobacteria MAE58620 And Human Homologous Protein ADH1B

Background: The production of algal toxins poses a serious threat to human and animal health,among which microcystins(MCs)are the most prominent.However,the biological function of MCs is still unclear.Although the toxicological targets of the toxin are mainly protein phosphatases(PPs)in human and mammalian,MCs emerged much earlier than the origin of metazoans,including mammals,and thus was not evolved by algae as a defense against metazoan feeding.Objective: Combined with the previous research results of our group and the international developments in recent years,this study selected cyanobacteria MAE58620 and human alcohol dehydrogenase(ADH)as the research object to explore its role in the biological function of MC-LR and discussed the potential impact of human ADH in the biotoxicity of MC-LR.Methods:1.Select Escherichia coli as the exogenous expression system,using the methods of genetic engineering,introduce 6×His tag and Kana resistance,and construct a recombinant expression plasmid of MAE58620,after culture expression,affinity chromatography was performed to obtain the protein.By setting the conditions of different temperatures,p H and metal ions,the enzymatic properties of recombinant protein were preliminarily explored.2.The biological function verification experiment of MC-LR was carried out using the successfully prepared recombinant protein MAE58620.Firstly,the biological information of MAE58620 was preliminarily explored.Secondly,Auto Dock Vina was used to conduct molecular docking experiments to study whether the two could interact.By setting different concentrations of MC-LR,the response and influence of MAE58620 to MC-LR were explored.Finally,the ITC was used to verify whether there was a combination between the two and obtain the corresponding binding thermodynamic parameters.3.Firstly,the homology of cyanobacteria MAE58620 and human ADH was compared by BLAST;Preliminary exploration of the bioinformatics properties of ADH1B;Molecular docking technology was used to explore whether the interaction between MC-LR and ADH1 B could occur.Secondly,the enzyme activity of ADH1 B at different concentrations of MC-LR was measured to verify whether the presence of MC-LR could cause changes in the enzyme activity of ADH1 B.Finally,ITC was used to verify whether there is a combination between the two and obtain the corresponding binding thermodynamic parameters.Results:1.In this study,the recombinant expression plasmid p ET-30a-ADH was successfully constructed,and stable transfected cells were obtained by synthesizing the coding gene of cyanobacteria MAE58620.The target protein obtained by affinity chromatography was detected by SDS-PAGE and Western Blot,there are obvious bands at 36 k Da and the anti-His antibody detected positive bands.It proved that the collection and purification of recombinant protein were successful.Through the preliminary exploration of the enzymatic properties of the protein,it can be seen that the optimal p H of the protein is 4.0,the optimal reaction temperature is 30°C,and the stability is better in the range of 25～35°C.2.In this study,through analyzing the nucleotide sequence of MAE58620,the amino acid sequence,physicochemical properties,protein hydrophobicity,transmembrane domain,phosphorylation site,secondary and tertiary structures were obtained.Firstly,Auto Dock Vina was used to preliminarily determine that the interaction between MC-LR and MAE58620 could occur.Secondly,the effect of MCLR on the enzyme activity of MAE58620 was studied by setting different concentrations of MC-LR,and it was known that MC-LR could improve the enzyme activity of MAE58620,and the catalytic efficiency increased with the increase of MCLR concentration,one-way ANOVA showed that the experimental results had a very significant effect(p<0.0001),.In addition,the ITC results also confirm the binding effect between the two and give a series of thermodynamic parameters.3.In this study,the amino acid sequences of cyanobacteria MAE58620 and human ADH were compared through the BLAST,and the homology of the two was confirmed,suggesting that MC-LR may have similar enzymatic activity patterns on the two.By analyzing the nucleotide sequence of ADH1 B,the amino acid sequence,physicochemical properties,protein hydrophobicity,trans-membrane domain,phosphorylation site,secondary structure and tertiary structure were obtained,and the bioinformatic properties of ADH1 B were preliminarily explored.Auto Dock Vina was used to conducting molecular docking to find out if there was a binding effect between MC-LR and ADH1 B.Different concentrations of MC-LR were set to study the effect of MC-LR on ADH1 B enzyme activity,it was known that the concentration of MC-LR could inhibit the enzyme activity of ADH1 B when it was higher than 2.5 μmol/L,and one-way ANOVA showed that the experimental results had a significant effect(p=0.0004).The thermodynamic parameters of the interaction between the two were obtained by ITC,and the results also confirmed the interaction relationship between the two.Conclusion: This study successfully obtained the exogenous recombinant protein cyanobacteria MAE58620 and preliminarily explored its enzymatic properties;found that MC-LR could increase the enzymatic activity of cyanobacteria MAE58620 when it was within the concentration range of 0.625～20 μmol/L,which verifies that cyanobacteria MAE58620 can be used as a molecular target and play a certain role in the biological function of MC-LR;found that MC-LR could inhibit the enzymatic activity of ADH1 B when it was in the concentration range of 5～20 μmol/L,which proved that ADH1 B could be used as a toxicological target and play a role in the toxicity of MC-LR in humans.