【摘要】 [目的]以小鼠基因组为模板克隆乳清酸蛋白(whey acidic protein gene,WAP)5′和3′调控区,并构建人乳铁蛋白(Human Lactoferrin,hLTF)乳腺特异性表达载体。[方法]以高保真PCR技术扩增WAP5′和WAP3′长度分别为3.5 kb和4.6 kb的DNA片段,并克隆测序,应用体外连接技术连接pcDNA3.0、WAP5′、WAP3′和hLTF,转化连接产物,以限制性内切酶酶切、PCR验证和琼脂糖电泳鉴定阳性克隆。[结果]内切酶酶切及PCR鉴定结果显示,WAP5′和WAP3′基因克隆成功,其hLTF乳腺特异性表达载体构建正确。[结论]成功克隆WAP5′和WAP3′调控区;成功构建了hLTF乳腺特异性表达载体pW2-hLTF。更多还原

【Abstract】 To clone whey acidic protein gene(WAP) 5′ regulatory region and 3′ regulatory region applied to mouse genome and construct the mammary glands-specific expressional vector of human lactoferrin(hLTF) gene,the 3.5kb and 4.6 kb regulatory region of the WAP5′ and WAP3′ are obtained with the high fidelity polymerase chain reaction(PCR),and inserted into pMD19-T vector subsequently.And then it was correctly verified that they were excised and inserted into pcDNA3.0 with hLTF in vitro to form pW2-hLTF.The pW2-hLTF was testified by endonuclease digestion,PCR and agarose electrophoresis.The result indicated that the enzyme digestion and PCR demonstrated that the WAP5′ and WAP3′ were cloned precisely and the construction of the mammary gland-specific vector was under anticipation.It was conclusion that the WAP regulatory region cloning and construction of mammary gland-specific expression vector pW2-hLTF were successfully established.更多还原