人胰岛素在乳腺细胞和组织中的高效生产

High-Level Production of Human Insulin in Mammary Cells and Glands

哺乳动物乳腺作为机体最大的分泌器官,被誉为是生物合成活性蛋白质和蛋白类药物的最有利场所。目前已有多个活性蛋白可以通过乳腺被高效的生产。然而,利用乳腺生产内分泌蛋白的效率却始终较低。乳腺中所含蛋白修饰酶种类的限制,对于内分泌蛋白的翻译后修饰存在一定的局限性。本研究以人胰岛素原基因为研究对象,通过基因工程技术将人胰岛素原基因中的两个内肽酶Ⅱ（endoproteasesⅡ,PC2）切割位点分别转变为乳腺可识别的内肽酶Ⅰ/Ⅲ（endoproteasesⅠ/Ⅲ,PC1/3）切割位点,构建乳腺特异表达载体,p BC1-663、p BC1-662和p BC1-792,并在乳腺细胞和小鼠模型中分别比较突变的人胰岛素原的表达对于胰岛素合成和分泌的影响。在细胞水平分析显示,表达突变型胰岛素原的细胞和培养液中的胰岛素含量显著高于对照组。进一步检测转基因小鼠泌乳期乳腺组织中胰岛素的含量显示,突变型人胰岛素原能够高效的在乳腺组织中合成人胰岛素,并高效的分泌到乳汁中。综上所述,将人胰岛素原蛋白内的蛋白酶切位点转变为乳腺内特有蛋白酶的酶解位点后,能够显著提高人胰岛素在乳腺细胞和组织中的合成和分泌效率。

Mammary gland as one of exocrine gland is important protein preparation factory in mammalian body. It was also bioreactor that was used to produce proteinic drugs. Recently, several active proteins were high-effective generation. However, the efficiency that produced endocrine protein, like insulin was lower. The enzymes in mammary that differed from endocrine organs might be barrier to post-translated modification of endocrine proteins. In present study, the codons of amino acid in human porinsulin gene that could be distinguished by PC2 were changed to PC1/3. Mutated and wide-type proinsulin genes were synthesized, and mammary gland-specific vectors, pBC1-663, pBC1-662 and pBC1-792 were constructed. Furthermore, the transfected cells and transgenic mice that expressed these vectors were achieved. Compared to control mammary ceils that expressed wide-type proinsulin, the high-level insulin was detected in cells that expressed mutated proinsulin, and the insulin were effectively secreted in culture medium. In addition, in mammary gland and milk of transgenic mice, expression of mutated proinsulin was also benefit to synthesize and secrete insulin. Taken together, Our results suggested that mutated proinsulin produce mature insulin in mammary gland more effective than wide-type.