Ⅱ型肺泡细胞特异表达SARS冠状病毒核衣壳蛋白转基因小鼠的建立  

Surfactant Protein A Promoter Directs the Expression of the SARS Coronavirus Nucleocapsid Protein in Alveolar TypeⅡ Cells of Transgenic Mice

作　　者：潘晓晨[1] 王友亮[1] 程萱[1] 侯宁[1] 崔芳[1] 时令[1] 刘伯华[2] 祝庆余[2] 秦鄂德[2] 杨晓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]军事医学科学院微生物流行病研究所,北京100071

出　　处：《生物技术通讯》2006年第4期516-519,共4页Letters in Biotechnology

基　　金：国家重点基础研究发展规划SARS专项基金项目(2003CB514131)

摘　　要：目的:建立Ⅱ型肺泡细胞特异表达SARS冠状病毒(SARS-CoV)核衣壳蛋白(N蛋白)的转基因小鼠。方法:用分子克隆的方法构建包括肺表面活性蛋白A(SP-A)启动子、SARS-CoVN蛋白基因、β-半乳糖苷酶(LacZ)报告基因和人生长激素(hGH)polyA的转基因载体pSP-A-N。以显微注射的方法将8.3kb的转基因片段引入小鼠受精卵。通过PCR、Southern印迹和LacZ染色检测子代小鼠中转基因的整合及表达。结果:共注射952枚受精卵,移植至42只假孕母小鼠的输卵管中发育,获得子代小鼠128只,经PCR、Southern印迹鉴定,其中11只小鼠基因组上整合有SARS-CoVN蛋白基因,整合率为8.6%。鉴定结果显示,11只转基因首建者小鼠中有1只表达外源基因并能正常传代。LacZ染色结果表明N蛋白基因在转基因小鼠Ⅱ型肺上皮细胞中特异表达。结论:成功构建了Ⅱ型肺泡细胞特异表达SARS-CoVN蛋白的转基因小鼠,为深入研究该基因的病理生理学效应奠定了基础。Objective： To generate transgenic mice that express severe acute respiratory syndrome associated coronavirus （SARS-CoV） nucleocapsid protein in alveolar type Ⅱ cells. Methods： An alveolar type Ⅱ cell specific transgenic construct containing the surfactant protein A（SP-A） promoter, the nucleocapsid protein gene, LacZ report gene and human growth hormone0aGH） polyA was generated. The 8.3 kb transgenic DNA fragments were introduced into fertilized eggs by microinjection. PCR, Southern blot and LacZ staining were used to examine the integration and the expression of the nucleocapsid gene. Results： The injected 952 eggs were implanted into the oviducts of 42 female mice. In the 128 offspring, there were 11 mice carrying the transgene identified by PCR and Southern blot analysis, the percentage ei^ciency of integration was 8.6%. One out of 11 transgenic founder mice expressed the foreign gene and was fertile. LacZ staining showed that the nucleocapsid gene was specifically expressed in alveolar type Ⅱ cells. Conclusion： A novel transgenic mouse that expresses SARS-CoV nucleocapsid gene specifically in alveolar type Ⅱ cells was established. It will benefit the studies on pathological function of nucleocapsid gene of SARS.

关 键 词：SARS冠状病毒 核衣壳蛋白 转基因小鼠 肺表面活性蛋白A 

分 类 号：Q78[生物学—分子生物学]