目的：用聚乙二醇（PEG）修饰集成干扰素突变体Ⅱ（IFN-Con-m2，IIFNm2），通过纯化获得新型修饰分子并对该分子进行抗胰蛋白酶水解稳定性及初步药代动力学研究。 方法：将mPEG20000定点偶联到IIFNm2的第86位Cys残基上，修饰后的产物经CM层析后，以SDS-PAGE考察其纯度，用WISH-VSV系统进行生物活性测定；在0.1%胰蛋白酶条件下考察体外抗酶解稳定性；并以SD大鼠进行初步药代动力学研究，绘制血药浓度-时间曲线。采用3P87软件进行数据拟合，分析药物动力学参数。 结果：干扰素修饰率约为50%，且绝大多数以单修饰体（mono-PEG- IIFNm2）形式存在；提纯后mono-PEG-IIFNm2 的纯度大于98%，比活性约为修饰前IIFNm2的1%。抗胰蛋白酶水解试验表明：30min后，IIFNm2抗病毒活性残留为8%，mono-PEG-IIFNm2为41%。初步药代动力学研究显示：IIFNm的消除半衰期为(1.57±0.34)h，mono-PEG-IIFNm2为(18.0±4.0)h。 结论：成功地偶联了PEG和IIFNm2，建立了mono-PEG-IIFNm2的纯化工艺，PEG修饰能增加IIFNm2的体外抗胰蛋白酶水解稳定性，并显著延长体内半衰期。
ecombinant interferon alpha (IFN-alpha) has been proven useful for treating a variety of human cancers and viral diseases. Because of its short circulating half-life IFN-alpha is administered to patients by daily or thrice weekly injection for optimal effectiveness. Recombinant consensus interferon mutant Ⅱ (IFN-Con-m2, IIFNm2) was a mutant from IFN-Con with a free cysteine residues at position 86.The protein could be modified with a 2 kDa-maleimide PEG and the mono-PEGylated proteins were purified by CM Sepharose Fast Flow column chromatography. Mono-PEGylated IIFNm2 could be separated from multi-PEGylated IIFNm2 and unmodified IIFNm2 by stepwise elution. After purification, the purity of mono-PEG-IIFNm2 was up to 98%, and the biological activity was more than 5.0×106IU/mg. The test of anti-trypsin digestion suggested that PEGylation could improve the ability of avoiding the trypsin digestion. Pharmacokinetic experiments in rats demonstrated that the circulating half-life of the PEGylated protein was up to 11.5-fold longer than that of IIFNm2. These data could demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting IIFNm2.
牛晓霞,周敏毅,刘金毅,孙超,钟茜,吴晓东. 聚乙二醇定点修饰集成干扰素突变体Ⅱ[J]. 中国生物工程杂志, 2008, 28(4): 17-20.
. Site-Specific PEGylation of Recombinant Consensus Interferon Mutant Ⅱ. China Biotechnology, 2008, 28(4): 17-20.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I4/17
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