人蛋白C转基因小鼠的建立及乳腺表达  

Establishment of Transgenic Mice for Expression of Human Protein C Gene in Mamma ry Gland

作　　者：赵宝全[1] 章金刚[1] 董朝辉[2] 贾莉玮[1] 沈永才[1] 吕茂民[1] 姚站馨[1] 李伟静[1] 孙曼霁[2] 

机构地区：[1]军事医学科学院输血研究所,北京100850 [2]军事医学科学院毒物药物研究所,北京100850

出　　处：《中国生物制品学杂志》2005年第4期291-293,共3页Chinese Journal of Biologicals

基　　金：军事医学科学院创新基金资助项目(020901)

摘　　要：目的为了获得乳腺定位表达人蛋白C(hPC)的转基因动物。方法采用分步克隆的方法,构建大鼠乳清酸蛋白(WAP)启动子调控的人蛋白C质粒,命名为pWPC。将该质粒系统鉴定后,用PvuⅠ+SmaⅠ双酶切,回收4·6kb的片段(WAP-hPC-PolyA)。通过显微注射技术,将其注入昆明小鼠受精卵雄原核内;共注受精卵810枚,存活640枚,移植给28只假孕母鼠,怀孕的18只母鼠共产仔81只。结果提取鼠尾基因组DNA进行PCR检测,34只阳性,再经Southernblot检测,其中20只整合阳性;将7只整合阳性母鼠传代,产仔7日后收集乳汁,进行ELISA检测,结果均有表达。结论已成功建立了人蛋白C转基因鼠乳腺生物反应器,为hPC乳腺表达研究奠定了基础。Objective To establish transgenic mice for the expression of human protein C gene in mamma ry gland. Methods A recombinant plasmid for expression of human protein C(hPC) under the control o f whey acid protein(WAP) promoter was constructed by stepwise cloning method and named as pWPC.The constructed plasmid was digested with PvuⅠ+SmaⅠ,and a 4.6 kb fragment(WAP-hPC-PolyA) was recovered and injected into male pro-nuclei of zygotes of Kunming mice by microinjection.Of the 810 zygotes injected,640 survi val ones were transplanted to 28 mice with pseudopregnancy.A total of 81 offspri ng were born from 18 pregnant mice.The genomic DNAs were extracted from the tail s of offspring for amplification by PCR. Results The genomic DNAs of hPC were amplified from 34 of the 81 offspring.Southern blot proved that the genomic DNAs of hPC were integrated to the genomic DNAs of 20 o f the 34 offspring.On the 7 th day after delivery,ELISA showed the express ion of hPC in the milk of 7 of the 20 mice with hPC DNA integrated. Conclusion The transgenic mice for expression of hPC in mammary gland was successfully esta blished.It laid a foundation of expression of hPC in mammary gland of mice.

关 键 词：人蛋白C 转基因小鼠 乳腺生物反应器 乳清酸蛋白 

分 类 号：TQ464.7[化学工程—制药化工] Q786[生物学—分子生物学]