CAJ | 학술논문

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(rRicin B)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-rRicin B原核表达系统,并通过IPTG诱导表达、三步洗涤、Ni2+-NTA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rRicin B与Hela细胞相互作用等.结果通过生物信息学预测到VVC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28.71 U/L;Hela细胞经FITC标记的rRicin B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.
목적 연구극륭표체적창상호균용세포소(vvC)중적중조비마독소B(rRicin B)공능모서,명학기공능,진일보천명창상호균용세포소적분자치병궤제.방법 생물신식학예측VVC중적공능모서,구건pET28a-rRicin B원핵표체계통,병통과IPTG유도표체、삼보세조、Ni2+-NTA주친화층석법순화、복성、ELISA검측화격광공취초현미경검측FITC표기적rRicin B여Hela세포상호작용등.결과 통과생물신식학예측도VVC중336～465위안기산서렬가능존재비마독소B련적공능모서,극륭표체저단모서후,기상대분자질량위20×103,경ELISA법검측복성후비마독소항원성위28.71 U/L;Hela세포경FITC표기적rRicin B작용후기세포막표면화포질균유록색형광산생.결론 VVC중존재구유유사천연비마독소B련공능적모서,능여세포막표면결합병진입포질,가능여창상호균용세포소막성공화세포독활성유관.
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-rRicin B.The recombinant plasmid was transformed into E.coli BL21 (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 rRicin 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 20×103.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-forming and the cytotoxicity of Vibrio vulnificus cytolysin.