Prokaryotic Expression Of Different Subtypes Of Serum Amyloid Protein A1 And The Analysis Of Their Anti-candida Albicans Activity

Serum amyloid A(SAA)is a family of serium proteins,which is highly conserved among various animals and tissues.The mature SAA protein is 12~14 kD,and consists of103~104 amino acids.The secondary structures of different SAAs have different oligomers.SAA is highly correlated with disease,and plays important roles in immunity.Studies have shown that SAA has an inhibitory effect on many Gram-negative bacteria,but the effect on fungi is not known.Therefore,exploring the inhibitory function of SAA on fungi will lay a good foundation for the study of the antibacterial mechanism of SAA or the new antibacterial mechanism,thus further providing theoretical guidance for the development of new medicine.In this study,recombinant human SAA1 protein was expressed in prokaryotic expression and purified by nickel column.The anti-microbial effect of SAA1 protein on Candida albicans SC5314 was also explored.The main research results are as follows.1.Construction of recombinant plasmid.According to gene cloning technology,three-subtype genes of the human SAA1 were obtained and ligated with vector pET-28 a to obtain recombinant SAA1 plasmids.2.Induction,purification and identification of the target protein.The recombinant vector was transformed into four bacterial strains of KS1000,BL21(DE3),Rosetta(DE3)and Rosetta gami2(DE3)to induce the expression of SAA1 proteins.The expressed proteins were examined by SDS-PAGE,and it was found that the expression of SAA1 was highest in BL21(DE3)under the same conditions.Therefore,BL21(DE3)was selected as the expression strain to induce the expression of SAA1-?,SAA1-? and SAA1-?.The cells were disrupted,proteins were dissolved,and purified by nickel column.Finally,the purity analysis of SAA1 proteins were carried out by SDS-PAGE,and the identification of proteins were confirmed by Western Blot.Results showed that the purified protein was the target protein and with high purity.3.Analysis of the anti-bacterial effect of SAA1.The different subtypes of purifiedSAA1 were diluted to a gradient concentration,and co-culture with C.albicans SC5314.It can be found that the number of colonies of Candida albicans SC5314 decreases as the concentration of SAA1 increased within a certain range,which indicated that the three subtypes of SAA1 had inhibitory effect on Candida albicans SC5314,which is dependent on concentration within a certain range.Moreover,the three subtypes of SAA1 showed different inhibition effects on Candida albicans SC5314.In this study,three subtypes of SAA1 proteins were expressed in prokaryotic cells,and their inhibitory effects on Candida albicans were studied.Subtype of protein with good bacteriostatic effect was obtained.This study provided a good application for SAA1 protein.