大连工业大学学报2025，Vol.44Issue(5)：313-319,7.DOI:10.19670/j.cnki.dlgydxxb.2025.0501
海胆Trf重组基因的原核表达及细菌结合特性与抑菌活性
Prokaryotic expression of recombinant Trf gene and bacterial binding characteristics and its bacteriostatic activity
摘要
Abstract
The vector pET-29b(+)-Trf-E1(292P)-His was constructed by site directed mutagenesis using seamless cloning technology,and the recombinant protein TRF-E1(292P)was expressed and purified in prokaryotic.TRF-E1(292P)is only in monomeric form and has a high expression level,with a purity of more than 90％.The polyclonal antibody was prepared by immunizing mice with purified TRF-E1(292P)as antigen.of which titer was 1∶51 200 determined by indirect ELISA.With the increasing concentrations of TRF-E1(292P),the binding abilities to Escherichia coli,Vibrio splendens,Pseudoalteromonas sp.and Bacillus cereus increased gradually determined by Western Blot,but no binding ability to Bacillus subtilis was detected.The saturated concentration of TRF-E1(292P)was determined by combining FITC labeled TRF-E1(292P)binding to Vibrio brilliant.It was found that when the concentration of FITC labeled TRF-E1(292P)was 50 μg/mL,the addition of unlabeled TRF-E1(292P)did not affect the binding of FITC labeled TRF-E1(292P)with Vibrio brilliant and the fluorescence intensity remained stable,indicating that TRF-E1(292P)was not replaced and the combination was irreversible.The result of antibacterial activity showed that treatment with 0.25 μg/μL TRF-E1(292P)for 12 h,the bacterial concentrations of Escherichia coli,Vibrio splendidus,Pseudoalteromonas sp.and Bacillus cereus were significantly reduced to 84.55％,73.10％,51.71％and 63.20％,respectively,but there was no significant inhibitory effect on the growth of Bacillus subtilis.The antibacterial effect exhibited a dose-dependent enhancement with the increasing concentrations of TRF-E1(292P)within 0 to 0.25 μg/μL.关键词
海胆Trf重组基因/定点突变/多克隆抗体/异硫氰酸荧光素/抑菌活性Key words
Trf recombinant gene/site directed mutation/polyclonal antibodies/fluorescein isothio-cyanate(FITC)/antibacterial activities分类
生物科学引用本文复制引用
毛佳妮,赵清雅,王晗..海胆Trf重组基因的原核表达及细菌结合特性与抑菌活性[J].大连工业大学学报,2025,44(5):313-319,7.基金项目
国家重点研发计划项目(2018YFC1406805). （2018YFC1406805）
