血清淀粉样P成分转基因小鼠模型的建立

目的:建立可表达血清淀粉样P成分(SAP)转基因小鼠模型,以研究SAP基因的功能。方法:采用基因重组技术将SAP基因插入到pCMV-Tag5A真核表达载体上。经BciVI限制性内切酶酶切后,琼脂糖凝胶电泳回收2.7 kb目的片断,受精卵原核显微注射法将其注入雄原核,制备转基因小鼠。采用PCR法在基因水平筛选SAP转基因小鼠阳性小鼠;免疫沉淀法结合免疫印迹法在蛋白水平检测SAP基因在PCR阳性转基因小鼠中的表达情况。结果:成功构建了pCMV-Tag5A/mSAP真核表达载体。经显微注射法将2.7 kb线性目的基因片断注射入受精卵雄原核,移植入代孕母鼠,所生127只小鼠,其中PCR鉴定获得10只转基因阳性小鼠。免疫沉淀法和免疫印记法随机检测83#♂founder、7#♀founder与C57 BL/6J小鼠回交F1代PCR阳性转基因小鼠血清中外源SAP基因蛋白水平的表达,发现均有表达。结论:该研究以C57BL/6J小鼠为研究对象,成功地产生能稳定遗传SAP基因的过表达转基因小鼠模型,它将有利于研究SAP基因在炎症、淀粉样沉淀、自身免疫系统中的作用。

Construction of Sermn Amyloid P Component Transgenic Mouse Model

The serum amyloid P component(SAP)transgenic mouse model was established in order to study the function of the SAP gene.The mouse SAP gene DNA fragment was amplied with PCR,then cloned into the pCMV-Tag5A expressed vector with the recombination DNA technique.After the BcivI restriction enzyme digestion,the 2.7 kb fragment was microinjected into male pronuclei of mouse zygotes.The new born mice were evaluated with multiplex polymerase chain reaction(PCR).Protein level expression of the foreign SAP gene was detected with immunoprecipitation(IP) & Western blotting(WB).The results indiated expression vector pCMV-Tag5A/mSAP was constructed successfully,and then microinjection embryos were transferred into oviducts of pseudopregnant female.127 mice were born and survived,10 of whom were verified to integrate the SAP gene in their genomic DNA.With immunoprecipitation & Western blotting,SAP protein was detected in the positive F1 transgenic mice.The conclusion was that C57BL/6J mice were used in the research and SAP gene was generation and SAP protein was expression.It was a valuable over-expression SAP gene disease model to study further the roles of SAP gene in the course of inflammation,amyliodosis and self-immunity system.