转基因小鼠乳腺表达人乳过氧化物酶的初步研究

A Preliminary Study on Transgenic Mouse Model of Human Lactoperoxidase

从人基因组PAC文库中筛选出人乳过氧化物酶(hLPO)基因,利用长片段PCR的方法获得hLPO基因5'端约3kb片段,通过酶切方法获得hLPO基因3'端约27kb片段,将这两部分拼接并克隆到乳腺特异性表达载体pBC1上,构建以山羊β-casein启动区指导的hLPO的转基因表达载体pBC1-hLPO。利用显微注射的方法获得28只F0代小鼠,经PCR检测和Southern杂交分析证实,有5只小鼠(4♂,1♀)为整合hLPO基因的转基因阳性小鼠,整合率为17.86%,整合拷贝数在1至5之间。利用SDS-PAGE凝胶电泳和Westernblot印迹分析F0、F1代共3只雌性转基因小鼠乳样,结果表明hLPO重组蛋白的特异条带不明显。

Human Lactoperoxidase gene （hLPO） has been cloned into pBC1 to construct pBC1-hLPO specialized expression vector in mammary glands, Human LPO gene was assembled by two gene fragments, which are 3 kilo base pairs in length and 27 kilo base pairs long re spectively, The shorter one was amplified by long-distance PCR and the other was cut down by restriction Agel, both based on the donor of PAC RP5-1171110 plasmid that contains hLPO gene, Here, We established the transgenic mouse model by microinjection into fertilized eggs, and then obtained 5 transgenic mice （4male, lfemale） out of 28 descendants, identified by PCR and Southern blot. The integration rate is 17,86%, and lactoperoxidase expression levels were determined by SDS-PAGE gel electrophoresis and Western blot. The hLPO recombinant protein band is not obvious as the antibodies are not very specific.