CAJ | 학술논문

目的构建重组人白细胞介素6-绿脓杆菌外毒素融合蛋白IL-6D24-PE40,以选择性杀伤高表达IL-6受体（IL-6R）的肿瘤细胞和白血病细胞。方法采用重叠延伸的基因融合技术将N-末端缺失24个氨基酸的重组人白细胞介素6（IL-6D24）cDNA与缺失细胞结合区的绿脓杆菌外毒素PE40基因进行融合，构建IL-6D24-PE40融合基因。利用HB101/pBV220表达系统，实现了IL-6D24-PE40融合蛋白在大肠杆菌中的高效表达，经MonoQ柱进行层析纯化，采用MTS法检测细胞毒活性。结果 IL-6D24-PE40融合蛋白在大肠杆菌中的表达水平达到40%～60%。包涵体蛋白经分离、复性及纯化得到纯度>95%的融合蛋白。Westernblot证明，纯化的融合蛋白与IL-6抗体及PEA抗体均发生特异性结合。细胞毒活性检测证实，IL-6D24-PE40融合蛋白能高度特异地选择性杀伤高表达IL-6R的U937细胞，ID50约为250ng/ml，而对不表达IL-6R的CEM细胞无杀伤作用。结论 IL-6D24-PE40融合蛋白能够选择性杀伤高表达IL-6R的靶细胞,有希望成为导向治疗高表达IL-6/IL-6R系统的某些白血病和其他肿瘤的新型导向药物。
목적 구건중조인백세포개소6-록농간균외독소융합단백IL-6D24-PE40,이선택성살상고표체IL-6수체（IL-6R）적종류세포화백혈병세포。방법 채용중첩연신적기인융합기술장N-말단결실24개안기산적중조인백세포개소6（IL-6D24）cDNA여결실세포결합구적록농간균외독소PE40기인진행융합，구건IL-6D24-PE40융합기인。이용HB101/pBV220표체계통，실현료IL-6D24-PE40융합단백재대장간균중적고효표체，경MonoQ주진행층석순화，채용MTS법검측세포독활성。결과 IL-6D24-PE40융합단백재대장간균중적표체수평체도40%～60%。포함체단백경분리、복성급순화득도순도>95%적융합단백。Westernblot증명，순화적융합단백여IL-6항체급PEA항체균발생특이성결합。세포독활성검측증실，IL-6D24-PE40융합단백능고도특이지선택성살상고표체IL-6R적U937세포，ID50약위250ng/ml，이대불표체IL-6R적CEM세포무살상작용。결론 IL-6D24-PE40융합단백능구선택성살상고표체IL-6R적파세포,유희망성위도향치료고표체IL-6/IL-6R계통적모사백혈병화기타종류적신형도향약물。
Objective To construct a human recombinant interleukin-6(IL-6)-Pseudomonas exotoxin IL-6D24-PE40 fusion protein and to analyze its ability to selectively kill cancer cells and leukemia cells expressing high levels of IL-6 receptors. Methods cDNA encoding the human IL-6 devoid of N-terminal 24 amino acids (IL-6D24) was ligated with DNA fragment coding the Pseudomonas exotoxin devoid of its cell recognition domain (PE40). The resultant genes were ligated into expression vector pBV220 and then transformed into E.coli HB101 cells. The expressed recombinant protein was purified to electrophoresis purity by Mono Q column. Its cytotoxicity was measured by the MTS colorimetric method using U937 cells as targets. Results Fusion protein IL-6D24-PE40 was expressed in inclusion bodies experiments at levels of 40%-60% of total proteins in bacterial cells. Western blotting showed that the purified product could react with IL-6 antibody and PEA antibody, respectively. IL-6D24-PE40 were specifically cytotoxic to U937 cells with ID50 of 250ng/ml and non-cytotoxic to IL-6 receptor-negative cell line CEM. Conclusion IL-6D24-PE40 fusion protein and derivatives can selectively kill the target cells which express high levels of IL-6 receptors.