天花粉蛋白的聚乙二醇定点修饰及其性质的初步研究

用聚乙二醇(PEG)定点修饰天花粉蛋白(TCS), 并比较了修饰前后TCS的生物活性、免疫原性及药代动力学等诸多性质. 选择TCS分子上可能的抗原决定簇位点KR173-174进行定点突变, 并将所构建的突变体TCS_KR173-174CG在大肠杆菌中表达及纯化. 通过该突变体第173位引入的半胱氨酸残基进行PEG定点修饰. 通过比较研究, 分析所构建的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, 研究结果表明, 所构建的突变型TCS(mTCS)的活性与野生型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、急性毒性以及药代动力学性质得到显著提升. 表明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.

Primary Studies on the Properties of Trichosanthin After Site-directed Mutagenesis and PEGylation

To construct a PEGylated trichosanthin(TCS) mutein by site-directed PEGylation and analyse its bioactivities, immunogenicity, acute toxicity and pharmacokinetics. By computer modeling, the site KR173-174 was identified as potential antigenic determinant of TCS. Then, a TCS mutein namely TCSKR173-174CG was constructed by site-directed mutagenesis and expressed in E.coli BL21(DE3). After that, the bioactivities, immunogenicity, acute toxicity and pharmacokinetics of the mutant TCS and PEGylated TCS were examined. It was shown that the bioactivities of the mutant TCS were similar to wild-type TCS(wTCS), but with much lower immunogenicity. The PEGylated TCS show a significant decrease in immunogenicity and acute toxicity, and a marked increase in plasma half-life when compared with unmodified TCS. But there is a problem of activity reduction. The site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.