Isolation,Purification And Prokaryotic Expression Of Flagellin Produced By Antagonistic Strain NJ13

A strain of Bacillus methylotrophicus was isolated from endophytes of ginseng.Previous studies have found that the crude protein of the sterile fermentation broth under 20% saturation ammonium sulfate precipitation has significant antagonistic effect on Cylindrocarpon destructans.Two antifungal active proteins were isolated by Sephadex G-25,and crude protein 2F was selected for subsequent experiments.The stability study found that the antifungal crude protein 2F was stable to ultraviolet light,and the activity was lost by half at pH 12.After treatment at high temperature(above 100?),chloroform and proteinase K,the antifungal activity was completely lost.The crude protein 2F was subjected to DEAE-Sepharose Fast Flow weak anion exchange chromatography and Superdex75 10/300 GL gel filtration chromatography to obtain a higher purity antifungal protein.After concentration,the antifungal protein concentration was determined to be 0.1026 mg/mL.SDS-PAGE results show a single band around 29 KDa.The amino acid sequence of the purified antifungal protein was identified by MALDI-TOF mass spectrometry and the coverage was 76% compared with the amino acid sequence of the flagellin FlgG derived from Bacillus velezensis UCMB5033(accession : CDG29557.1).The flagellin FlgG has an isoelectric point of 4.97 and a molecular weight of 27 kDa,which is almost identical to the apparent molecular weight in the SDS-PAGE gel.It is preliminarily determined that the antifungal protein isolated and purified in this experiment is a flagellin-like protein.A specific primer G1 was designed based on the gene sequence of flagellin FlgG(accession : CDG29557.1),and a 777 bp gene sequence was cloned from the biocontrol bacteria NJ13 genomic DNA,which was the same size as the flagellin gene sequence.The cloned gene sequence was ligated to the PMD18-T vector,and after sequencing,the cloned gene was found to be 10 bases different from the flagellin FlgG gene sequence.However,the amino acid sequence encoded by the cloned gene is identical to the amino acid sequence of the flagellin FlgG.Therefore,it is considered that the cloned gene sequence is the flagellin gene and is subjected to bioinformatics analysis.The target gene fragment was recovere by double enzyme digestion and ligated to the expression vector PET-28a(+).After confirming by restriction enzyme identificatio,PCR and sequence analysis,the expression vector was transformed into E.coli BL21(DE3)for induction expression.The optimal conditions for expression of the target gene were determined by screening the inducer IPTG concentration and culture temperature.The cells were sonicated and the recombinant protein was collected for antifungal activity detection.Through this study,it is expected that a single component of the antifungal protein can be isolated and purified,and the heterologous expression of the antifungal protein can be achieved by genetic engineering.It lays a foundation for the discovery of genes related to antifungal diseases in the later stage,and then realizes the improvement of biocontrol bacteria,which is of great significance for the development of biological pesticides.