骨骼肌特异表达卵清蛋白(OVA)抗原转基因小鼠构建  

Generation of transgenic mice expressing neo-autoantigen OVA exclusively in skeletal muscle

作　　者：黄涛[1] 丁茂超[1,2] 史丹丹[1] 谷瑞彩 肖将尉 郭梦霞[1] 李俊桦[1] 廖华[1] 

机构地区：[1]南方医科大学基础医学院解剖学教研室广东省组织构建与检测重点实验室,广州510515 [2]温州医科大学人体解剖学教研室,325035

出　　处：《免疫学杂志》2017年第8期733-736,共4页Immunological Journal

基　　金：国家自然科学基金(81371924;81572102);广东省自然科学基金(2014A030313276)

摘　　要：目的利用PiggyBAC转座酶系统及受精卵注射法,将MCK3E-OVA-pA随机插入小鼠基因组,获得骨骼肌特异表达卵清蛋白(OVA)的转基因小鼠(Muscle-OVA Tg)。方法通过体外转录,获得PiggyBAC mRNA;将PiggyBAC mRNA和含有鸡全长OVA片段、骨骼肌肌酸激酶启动子(MCK)、H-2K^b引导序列、H-2Db跨膜序列的质粒显微注射至C57BL/6J小鼠受精卵中,获得转基因阳性首建鼠。将Muscle-OVA Tg鼠与野生B6小鼠杂交建系。PCR鉴定小鼠基因型;Westernblot检测小鼠各组织中OVA蛋白表达以验证其组织特异性。结果PCR检测表明,Muscle-OVA Tg的肌组织表达OVA mRNA;Western blot进一步证实Muscle-OVA Tg小鼠骨骼肌组织特异的OVA蛋白表达,其他组织/器官,如心、脾、肺、肾组织OVA表达均呈阴性。结论利用PiggyBAC转座酶系统成功构建骨骼肌特异表达鸡OVA抗原的转基因小鼠,为相关的骨骼肌免疫反应研究提供了有价值的动物模型。To construct a transgenic mouse model expressing neo-autoantigen OVA exclusively in skeletal muscle, we inserted MCK3E-OVA-pA into mouse genome randomly by PiggyBAC translocation enzyme system and fertilized egg injections. PiggyBAC m RNA was obtained by transcription in vitro, then the PiggyBAC mRNA and a plasmid containing chicken OVA fragments, skeletal muscle creatine kinase promoter（MCK）, boot sequence H-2K^b and transmembrane sequence H-2Db were microinjected into the fertilized eggs of C57BL/6 J mice to get founder mice. Tg mice was crossed with wild B6 mice. PCR and Western blot detections confirmed the expression of OVA in skeletal muscle is exclusive. PCR analysis demonstrated that the OVA mRNA was expressed in muscle tissue of Muscle-OVA Tg mice, while Western blot confirmed that OVA protein was expressed exclusively in skeletal muscle, other tissue/organ, such as the heart, spleen, lung and kidney tissues were negative. Taken together, a muscle-OVA Tg mouse model has been generated by using PiggyBAC translocation enzyme system, which could be used for study of immune response of skeletal muscle.

关 键 词：卵清蛋白 PIGGYBAC 转基因 骨骼肌 

分 类 号：R392[医药卫生—免疫学]