蚕蛹粉中有助于rhGM-CSF口服吸收的蛋白成分的分离纯化及鉴定
he purification and identification of protein from pupa powder which can help rhGM-CSF absorption by oral administration
蛋白药物的口服一直是世界性难题。实验中以蚕蛹粉作为载药材料，口服给药实验表明，蚕蛹粉能够一定程度上帮助rhGM-CSF的口服吸收，且在给药40 min后血药浓度达到最大值。为了从蚕蛹粉中寻找帮助rhGM-CSF口服吸收的有效蛋白成分，本文先通过匀浆和离心方法将蚕蛹粉分为可溶组分和不可溶组分，即上清和沉淀两部分。口服实验分析表明上清与rhGM-CSF药物混合物的生物利用度是沉淀与rhGM-CSF药物混合物的20倍。再利用硫酸铵分级沉淀法和10 kDa，30 kDa和100 kDa超滤膜分离上清中的蛋白，分别获得10-20 kDa蛋白，30 kDa蛋白，80 kDa蛋白。口服实验分析表明，10-20 kDa蛋白能够促进rhGM-CSF口服吸收，以仅口服rhGM-CSF药物为参比制剂，10-20 kDa蛋白与rhGM-CSF口服剂型的相对生物利用度是110%。10-20 kDa蛋白与rhGM-CSF药物不同配比比例的实验表明10-20 kDa蛋白含量越高，这种促进口服吸收效应越强，两者呈正相关性。Resource Q阴离子柱交换以及MALDI TOF MS和液质联用型LTQ分析10-20 kDa分子量大小范围内的蛋白，质谱分析结果表明10-20 kDa蛋白是ubiquitin，chymotrypsin inhibitor，Cationic peptide CP8 precursor，Kazal-type proteinase inhibitor和RecName: Full=Chymotrypsin inhibitor SCI-II。除ubiquitin外其余的蛋白均为丝氨酸蛋白酶抑制因子，能够阻止蛋白的水解作用，这可能是10-20 kDa蛋白能够促进rhGM-CSF口服吸收的原因。
he oral delivery of protein drug is a worldwide problem. In experiment, pupa powder were orally administered to Km as drug loading material mixed rhGM-CSF drugs. Blood sample was collectednd and serum was used to detect the concentration of rhGM-CSF by using rhGM-CSF ELISA Kit.In this paper, we extracted a certain type of proteins from the pupa powder，and as drug loading material to manufacture a novel oral dosage form of the rhGM-CSF to improve the oral bioavailability of the rhGM-CSF. The result indicated, the concentration of rhGM-CSF in bloodstream was highest during the experimental group, mixture of pupa powder and rhGM-CSF drug, at 40 min post oral administration. After, we divided pupa powder into two parts of the supernatant and precipitant, through homogenizer and centrifugation. The result of animal experiment and ELISA suggested the bioavailability of mixture of supernatant and rhGM-CSF was 20 times the mixture of precipitate and rhGM-CSF. With ammonium sulfate precipitation method and ultrafiltration, 80 kDa proteins, 30 kDa proteins and 10-20 kDa proteins were separated from the supernatant. The result of animal experiment and ELISA maintained the bioavailability of mixture of 10-20 kDa proteins and rhGM-CSF was highest, and was 110 times of the bioavailability of oral rhGM-CSF without any drug loading material. The experimental results of different mixing ratio of rhGM-CSF drugs and 10-20 kDa protein suggested, the effect of 10-20 kDa protein was relative to the mixing ratio, and the mixing ratio was larger the effect was better. It was positive correlation.The analysis of MALDI TOF MS and LTQ MS showed that the protein of 10-20 kDa proteins were ubiquitin, chymotrypsin inhibitor, Cationic peptide CP8 precursor, Kazal-type proteinase inhibitor and RecName: Full=Chymotrypsin inhibitor SCI-II. In addition to ubiquitin, the rest of the proteins are serine protease inhibitor, which can prevernt protease hydrolysis. This may be the reason why 10-20 kDa proteins can help the rhGM-CSF oral administration.
王鉴、舒特俊、张耀洲、陈剑清、李伟杰
药学生物科学理论、生物科学方法生物化学
生物化学10-20 kDa蛋白口服生物利用度rhGM-CSF分离纯化MALDI TOF MS液质联用LTQ
Silkworm powder10-20 kDa proteinsOral bioavailabilityrhGM-CSFSeparation and purificationMALDI TOF MSLTQ-MS
王鉴,舒特俊,张耀洲,陈剑清,李伟杰.蚕蛹粉中有助于rhGM-CSF口服吸收的蛋白成分的分离纯化及鉴定[EB/OL].(2012-12-27)[2025-08-18].http://www.paper.edu.cn/releasepaper/content/201212-989.点此复制
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