牛乳铁蛋白肽转基因小鼠的构建

目的:探讨牛乳铁蛋白肽在转基因鼠乳汁中的表达及其抑菌活性。方法:将实验室构建并保存的包含山羊β-酪蛋白基因启动子和牛乳铁蛋白肽基因的乳腺特异表达载体PI-bcp-LfcinB,用Xho I和Nru I双酶切,得到含有全部表达盒的显微注射DNA片段,采用常规显微注射技术获得转基因小鼠。并通过对泌乳期转基因雌鼠乳腺组织的RT-PCR检测,确定了牛乳铁蛋白肽在mRNA水平的表达,同时利用琼脂板扩散法检测了转基因鼠乳汁中表达产物的抑菌活性。结果:获得了牛乳铁蛋白肽转基因小鼠,且转基因小鼠乳汁中能够表达具有抑菌活性的牛乳铁蛋白肽。结论:通过转基因动物乳腺可以获得具有生物活性的牛乳铁蛋白肽,为进一步研究抗菌肽转基因牛、培育抗乳房炎奶牛新品种以及通过建立转基因动物生物反应器进行抗菌肽的大量生产奠定了基础。

Construction of Bovine Lactoferricine Transgenic Mice

Purpose: To investigate the expression of bovine lactoferricine in mammary gland of the transgenic mice and the antibacterial activity. Methods: Mammary gland expression vector PI-bcp-LfcinB containing the goat β-casein promoter and bovine lactoferricine gene was digesting with Xho I and Nru I. DNA fragment containing the expression elements was gained for microinjection. About 500 fertilized eggs of mice were injected using classic microinjection technique,and about 430 living eggs were transplanted into 13 pseudopregnant mice,among which 6 pregnant mice produced 13 offspring.These offspring were identified by PCR,1 mice was bovine lacteferricine transgenic mice.Target gene can be transferred by the transgenic mice,which was proved by PCR. The expression of bovine lactoferricine of transgene mice was detected by RT-PCR and Kirby-Bauer. Results: These tests showed that the mammary-tissue-specific expression vector driven by the goat beta-casein gene promoter could efficiently direct the expression of bovine lactoferricine in mice milk. And the expressed product exhibited evident bactericidal activity to Escherichia coli. Conclusion: It is feasible to get bioactive bovine lactoferricine through the mammary gland of animal and it provides a basis for disease-resistant animal breeding and production of antibacterial peptides through transgenic animals.