Wei Y, Yarus S, Greenberg N M, Whitsett J, Rosen J M
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Transgenic Res. 1995 Jul;4(4):232-40. doi: 10.1007/BF01969116.
Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3' untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expression levels were greater than those of of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12-14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12-14 kDa is a processed form of SP-C.
呼吸窘迫综合征（RDS）由肺表面活性物质缺乏引起，在美国每年影响65000名婴儿。表面活性物质替代疗法可降低与RDS相关的发病率和死亡率。人表面活性蛋白C（SP-C）是肺表面活性物质的重要组成部分。为了生产人SP-C，构建了一种利用大鼠乳清酸性蛋白（WAP）启动子和3'非翻译区将人SP-C基因表达靶向转基因小鼠乳腺的载体。在所分析的所有转基因品系中均检测到WAP/SP-C mRNA表达。SP-C以拷贝数依赖和整合位点独立的方式表达，表达水平为内源性小鼠WAP mRNA的0.01%至36.0%，且WAP/SP-C mRNA表达水平高于内源性小鼠肺SP-C mRNA水平。RNA水平的表达在乳腺中具有特异性，并且在乳腺发育过程中与内源性WAP表达模式平行。使用抗SP-C多克隆抗体对全脂牛奶进行的蛋白质印迹法证实了泌乳乳腺中SP-C蛋白的表达和分泌。分子量为22 kDa和12 - 14 kDa的免疫反应性蛋白仅出现在转基因牛奶中。22 kDa的蛋白代表前体蛋白，12 - 14 kDa是SP-C的加工形式。
