创伤弧菌溶细胞素蓖麻毒素B活性模序的预测、克隆表达和功能鉴定

Pre, diction, cloning, expression and identification of the functional motif of the recombinant Ricin Bin Vibrio vulnificus cytolysin

目的研究克隆表达的创伤弧菌溶细胞素（VVC）中的重组蓖麻毒素B（rRicinB）功能模序，明确其功能，进一步阐明创伤弧菌溶细胞素的分子致病机制。方法生物信息学预测VVC中的功能模序，构建pET28a-rRicinB原核表达系统，并通过IPTG诱导表达、三步洗涤、Ni24NTA柱亲和层析法纯化、复性、ELISA检测和激光共聚焦显微镜检测FITC标记的rRicinB与Hela细胞相互作用等。结果通过生物信息学预测到YVC中336～465位氨基酸序列可能存在蓖麻毒素B链的功能模序，克隆表达这段模序后，其相对分子质量为20×103，经ELISA法检测复性后蓖麻毒素抗原性为28．71U／L；Hela细胞经FITC标记的rRicinB作用后其细胞膜表面和胞质均有绿色荧光产生。结论VVC中存在具有类似天然蓖麻毒素B链功能的模序，能与细胞膜表面结合并进入胞质，可能与创伤弧菌溶细胞素膜成孔和细胞毒活性有关。

Objective To determine the functional motif of the recombinant Ricin B （rRicin B） in Vibrio vulnificus cytolysin （VVC） and understand its molecule pathogenic mechanism. Methods The motif of VVC was predicted through bioinformatics analysis and cloned into a procaryotic expression vector pET28a-rRiein B. The recombinant plasmid was transformed into E. coli BI221 （ DE3 ） and induced by IPTG to express rRicin B. The expressed protein was further analyzed by SDS-PAGE and purified by Ni2^-NTA agarose. Renaturation of the rRiein B were also carried out for further analysis. ELISA assay and confocal microscope was applied to identify the activity of the rRicin B on human Hela cells. Results Ricin B motif located in the 336-465 amino acids of Vibrio vulnificus cytolysin with a relative molecular weight of 20x103. The result of ELISA showed that the antigenicity of rRicin B was 28.71 U/L after renaturation. FITC labeled rRicin B could bind to the cell membrane and enter the cytoplasm of human Hela cells. Conclusion The Ricin B motif in Vibrio vulnificus cytolysin bearing the similar ability with the natural Ricin B can bind to the cell membrane and enter the cytoplasm. This feature may play an important role in the activity of pore-form- ing and the cytotoxicity of Vibrio vulnificus cytolysin.