Zhang Yu-Qing, Ma Yan, Xia Yun-Yue, Shen Wei-De, Mao Jian-Ping, Zha Xin-Min, Shirai Koji, Kiguchi Kenji
Silk Biotechnology Lab., School of Life Science, Soochow University, Dushuhu Higher Education Town, Suzhou 215123, People's Republic of China.
J Biomed Mater Res B Appl Biomater. 2006 Nov;79(2):275-83. doi: 10.1002/jbm.b.30539.
The regenerated liquid silk fibroin with an average molecular mass of about 60 kDa consists of 18 kinds of amino acids containing approximately 10% of polar amino acids with hydroxyl and amino groups such as serine and lysine. The liquid silk fibroin is coupled covalently with insulin molecules through these strongly polar side groups by using glutaraldehyde. The physicochemical properties of the silk fibroin-insulin (SF-Ins) bioconjugates were investigated by enzyme-linked immunosorbent assay for the quantitative measurement of insulin. The biological activities of the insulin bioconjugates were characterized in vitro and in vivo. The SF-Ins constructs obtained by 5 h of covalent crosslinking showed much higher recovery (about 70%) and in vitro stability in human serum than bovine serum albumin-insulin (BSA-Ins) derivatives. The results in human serum indicated that the half-life in vitro of the biosynthesized SF-Ins derivatives was 2.1 and 1.7 times more than that of BSA-Ins conjugates and native insulin, respectively. The immunogenicity of the regenerated silk fibroin and the antigenicity of silk fibroin-modified insulin were not observed in both rabbits and rats. The pharmacological activity of the SF-Ins bioconjugates in diabetic rats evidently lengthened and was about 3.5 times as long as that of the native insulin, nearly 21 h. The bioconjugation of insulin with the regenerated silk fibroin greatly improved its physicochemical and biological stability.
平均分子量约为60 kDa的再生液态丝素蛋白由18种氨基酸组成，其中含有约10%的带有羟基和氨基的极性氨基酸，如丝氨酸和赖氨酸。液态丝素蛋白通过戊二醛利用这些强极性侧基与胰岛素分子共价偶联。通过酶联免疫吸附测定法对丝素蛋白 - 胰岛素（SF - Ins）生物共轭物的理化性质进行研究，以定量测定胰岛素。胰岛素生物共轭物的生物活性在体外和体内进行了表征。通过5小时共价交联获得的SF - Ins构建体在人血清中的回收率（约70%）和体外稳定性比牛血清白蛋白 - 胰岛素（BSA - Ins）衍生物高得多。人血清中的结果表明，生物合成的SF - Ins衍生物在体外的半衰期分别比BSA - Ins共轭物和天然胰岛素长2.1倍和1.7倍。在兔子和大鼠中均未观察到再生丝素蛋白的免疫原性和丝素蛋白修饰胰岛素的抗原性。SF - Ins生物共轭物在糖尿病大鼠中的药理活性明显延长，约为天然胰岛素的3.5倍，接近21小时。胰岛素与再生丝素蛋白的生物共轭极大地提高了其理化和生物稳定性。
