Cloning,Expression,Purification And Application Of Glycosylation Detection Of Antheraea Pernyi ApMBL-EGFP Fusion Protein

The ability of lectins to specifically recognize sugars makes them widely used in glycosylation studies.Biotin-avidin-system(BAS)is widely used in lectin research glycosylation technology.The principle of the biotin-avidin system is to first incubate the glycoprotein with the biotinylated lectin,then with the avidin-enzyme conjugate,and finally use the substrate to detect the enzyme bound to the avidin.The analysis of glycoproteins is achieved indirectly based on the substrate signal.The system has cumbersome steps,high cost,and many reaction layers,which affect its specificity.Therefore,in this study,a lectin and fluorescent fusion protein was prepared,and the glycoprotein was directly detected by the fluorescent signal on the lectin,which greatly simplified the steps,reduced the production cost,reduced the reaction level,and improved its specificity.In this study,the c DNA sequence of the antheraea pernyi lectin Ap MBL was cloned and analyzed by bioinformatics.The results showed that Ap MBL protein encoding 327 amino acids,and the first 23 positions were its signal peptide region.It is predicted to have a molecular weight of 37.5KDa and an isoelectric point of 5.47.Ap MBL protein has high homology with VIP36 protein and contains an L-type lectin domain and a transmembrane region.β-sheet(extended strand)and random coil are its main secondary structure elements.The lectin Ap MBL gene and the EGFP gene were spliced together through the Linker sequence to form the fusion gene Ap MBL-EGFP,and the rationality of its design was tested by bioinformatics analysis.The results showed that the molecular weight of the Ap MBL-EGFP fusion protein was 67 KDa and the isoelectric point was 6.41.The addition of Linker sequence made the structure and function of the Ap MBL protein and EGFP protein independent,and had strong hydrophilicity.The two fragments of Ap MBL and EGFP were ligated by fusion PCR,and then ligated to the p ET-28(a+)expression vector,and transformed into E.coli BL21(DE3).The expression was induced with IPTG,and then the Ap MBL-EGFP fusion protein was purified by nickel column affinity chromatography through the N-terminal His tag of the fusion protein.After SDS-PAGE electrophoresis,Coomassie brilliant blue staining and Western blot analysis,the results showed that the Ap MBL-EGFP fusion protein was successfully expressed and purified in the E.coli expression system.The fluorescence activity,agglutination activity and sugar-binding activity of Ap MBL-EGFP fusion protein were detected by fluorescence spectrum analysis,agglutination bacteria experiment and enzyme-linked immunosorbent assay(ELISA).The results show that the Ap MBL-EGFP fusion protein has a fluorescence spectrum consistent with the trend of EGFP.It has high agglutination activity against Salmonella enterica,Escherichia coli and Bacillus methylotrophic bacteria.It has a certain binding activity with lipopolysaccharide,peptidoglycan,mannan and maltotriose,and it is concentration-dependent.Finally,the purified Ap MBL-EGFP fusion protein was used for glycoprotein dot western blot analysis,The results show that the application of this protein can effectively detect glycoproteins in dot blot analysis,and compared with the biotin-avidin system,it has the advantages of simple experimental steps,low production cost,and no additional color reagents.Has great potential in lectin immunoassays.