發(fā)布時間：2019-04-21 17:14

【摘要】：采用分子質(zhì)量為40k Da的馬來酰亞胺聚乙二醇(m PEG-MAL),對重組人睫狀神經(jīng)營養(yǎng)因子突變體CNTF-C17的第17位半胱氨酸巰基進行定點修飾,通過離子交換層析獲得單修飾產(chǎn)物Mono-PEG-CNTF-C17,并對其結(jié)構(gòu)及體內(nèi)外活性進行評價。實驗結(jié)果表明,在p H 7.5的Tris-HCl緩沖液體中,蛋白質(zhì)與修飾劑的為1∶3,4℃下反應(yīng)12h,修飾率可達到90%以上,修飾混合物通過一步陰離子交換層析可獲得純度98%以上的單修飾產(chǎn)物。熒光光譜(FL)及圓二色(CD)圖譜顯示Mono-PEG-CNTF-C17與原蛋白二、三級結(jié)構(gòu)一致。TF-1.CN5a.1細胞增殖活性檢測表明,MonoPEG-CNTF-C17的比活達到了6.51×105IU/mg,體內(nèi)循環(huán)半衰期相對原蛋白顯著提高了30.3倍。該研究可為開發(fā)CNTF長效產(chǎn)品提供基礎(chǔ)。
[Abstract]:The 17th cysteine sulfhydryl group of recombinant human ciliary neurotrophic factor mutant CNTF-C17 was modified by maleimide polyethylene glycol (m PEG-MAL) with molecular weight of 40 k Da. The single modified product Mono-PEG-CNTF-C17, was obtained by ion exchange chromatography and its structure and activity in vitro and in vivo were evaluated. The results showed that in pH 7.5 Tris-HCl buffer solution, the ratio of protein and modifier was 1? 3, and the modification rate was more than 90% at 4 鈩，

本文編號：2462396