中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2008年
20期
1433-1436
,共4页
安群星%雷迎峰%穆士杰%张献清%陈蕤%夏爱军%陈晨%易静%吴原茹%余瑞%徐志凯
安群星%雷迎峰%穆士傑%張獻清%陳蕤%夏愛軍%陳晨%易靜%吳原茹%餘瑞%徐誌凱
안군성%뢰영봉%목사걸%장헌청%진유%하애군%진신%역정%오원여%여서%서지개
天花粉素%聚乙烯二醇类%药代动力学%HIV
天花粉素%聚乙烯二醇類%藥代動力學%HIV
천화분소%취을희이순류%약대동역학%HIV
Trichosanthin%Polyethylene glycols%Pharmaeokinetics%HIV
目的 对天花粉蛋白(TCS)进行定点突变、聚乙二醇(PEG)修饰,并将修饰前后TCS的诸多性质加以比较.方法 选择TCS分子上可能的抗原决定簇位点YFF81-83进行定点突变,并将所构建的突变体TCSYFF81-83ACS在大肠杆菌中表达及纯化.随后通过该突变体第82位刚引入的半胱氨酸残基进行PEG定点修饰.通过比较研究,分析所构建PEG修饰型TCS的DNA酶活性、致核糖体失活活性、免疫原性、急性毒性以及药代动力学性质.结果 所构建的突变型TCS其活性与野生型TCS(wTCS)活性几乎相当,而免疫原性已显著降低.PEG修饰型TCS虽有活性下降,但其免疫原性比wTCS低(P＜0.05),急性毒性比wTCS低,LD50值约为wTCS的1.8倍(P＜0.05),血液内平均滞留时间和半衰期明显长于wTCS(P＜0.05).结论 通过基因工程及化学修饰方法改造TCS是可行的,所构建的突变型及PEG修饰型TCS值得进一步研究.
目的 對天花粉蛋白(TCS)進行定點突變、聚乙二醇(PEG)脩飾,併將脩飾前後TCS的諸多性質加以比較.方法 選擇TCS分子上可能的抗原決定簇位點YFF81-83進行定點突變,併將所構建的突變體TCSYFF81-83ACS在大腸桿菌中錶達及純化.隨後通過該突變體第82位剛引入的半胱氨痠殘基進行PEG定點脩飾.通過比較研究,分析所構建PEG脩飾型TCS的DNA酶活性、緻覈糖體失活活性、免疫原性、急性毒性以及藥代動力學性質.結果 所構建的突變型TCS其活性與野生型TCS(wTCS)活性幾乎相噹,而免疫原性已顯著降低.PEG脩飾型TCS雖有活性下降,但其免疫原性比wTCS低(P＜0.05),急性毒性比wTCS低,LD50值約為wTCS的1.8倍(P＜0.05),血液內平均滯留時間和半衰期明顯長于wTCS(P＜0.05).結論 通過基因工程及化學脩飾方法改造TCS是可行的,所構建的突變型及PEG脩飾型TCS值得進一步研究.
목적 대천화분단백(TCS)진행정점돌변、취을이순(PEG)수식,병장수식전후TCS적제다성질가이비교.방법 선택TCS분자상가능적항원결정족위점YFF81-83진행정점돌변,병장소구건적돌변체TCSYFF81-83ACS재대장간균중표체급순화.수후통과해돌변체제82위강인입적반광안산잔기진행PEG정점수식.통과비교연구,분석소구건PEG수식형TCS적DNA매활성、치핵당체실활활성、면역원성、급성독성이급약대동역학성질.결과 소구건적돌변형TCS기활성여야생형TCS(wTCS)활성궤호상당,이면역원성이현저강저.PEG수식형TCS수유활성하강,단기면역원성비wTCS저(P＜0.05),급성독성비wTCS저,LD50치약위wTCS적1.8배(P＜0.05),혈액내평균체류시간화반쇠기명현장우wTCS(P＜0.05).결론 통과기인공정급화학수식방법개조TCS시가행적,소구건적돌변형급PEG수식형TCS치득진일보연구.
Objective To construct PEGylated trichosanthin(TCS)mutein and analyze its bioactivities,immunogenicity,acute toxicity,and pharmacokineties.Methods The potential antigenic determinant site YFF81-83 in the molecule of TCS was selected to undergo site-directed mutagenesis.Thus,a TCS mutein named TCSYFF81-83ACS was constructed and expressed in Escherichia coli of the line BL21 (DE3).Wild TCS(wTCS),TCSYFF81-83ACS,and PEGylated TCSYFF81-83ACS(PEG-TCSYFF81-83ACS)of different concentrations were incubated with the supercoiled plasmid pUCl9 to detect the DNAse activity,mixed with rabbit reticulocyte lysate to detect the ribosome inactivation activity,subcutaneously injected into 6 mice respectively to measure the serum IgG and IgE levels,intravenously injected into mice to observe the toxicity,and intravenously injected into SD rats to observe its-plasma half-life.Results The DNAse activity of the PEG-TCSYFF81-83ACS was similar to that of the wTCS.The ribosome inactivation activity of the PEG-TCSYFF81-83ACS was 1/9-1/8 of that of the wTCS(P＜0.05).The serum IgE and IgG levels of the PEG-TCSYFF81-83ACS were both significantly lower than those of the wTCS(both P＜0.05).The LD50 of the PEG-TCSYFF81-83ACS was 1.8 times that of the wTCS(P＜0.05).The mean residence time and plasma half-life of the PEG-TCSYFFF81-83ACS were significantly increased and its plasma clearance was significantly decreased(all P＜0.05).Conclusion Site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.
