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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。
Purpose To obtain a transgenic animal that has mammary gland localization of human protein C (hPC). Methods The stepwise cloning method was used to construct the human protein C plasmid regulated by the rat whey acid protein (WAP) promoter and named pWPC. After identification of the plasmid system, a 4.6 kb fragment (WAP-hPC-PolyA) was recovered by double digestion with PvuI + SmaI. A total of 810 fertilized eggs were injected into the male pronucleus of the fertilized ovum of Kunming mice via microsurgical injection. A total of 810 fertilized eggs were co-injected, and 640 surviving mice were transplanted to 28 fake pregnant rats and 81 pregnant rats. Results Genomic DNA was extracted from the tail of the mice for PCR. Twenty-four of the samples were positive by Southern blot analysis, of which 20 were positive for integration. Seven integrated positive mice were passaged and collected for lactation after 7 days of birth. ELISA was performed and the results were expressed. Conclusion Human protein C transgenic mouse mammary gland bioreactor has been successfully established, which lays the foundation for the study of hPC mammary gland expression.