抗病毒基因MxA真核表达载体的构建及在鸡细胞中的表达

Construction of Eukaryotic Expression Vector Containing Antiviral MxA Gene and Its Expression in Chicken Cells

将人抗病毒蛋白基因MxA与携带增强型绿色荧光蛋白(EGFP)基因的真核表达质粒重组后构建MxA基因真核表达载体pEGFP-C1-MxA。经PCR和酶切方法鉴定后,重组质粒在脂质体介导下转染鸡成纤维细胞和睾丸组织原代细胞,通过荧光观察,RT-PCR及细胞免疫组化检测目的基因的表达。结果表明,MxA基因片段已经被克隆到pEGFP-C1表达载体,成功构建了MxA基因真核表达载体pEGFP-C1-MxA。经该重组质粒转染后的鸡细胞的胞质中呈现颗粒状分布的绿色荧光,RT-PCR扩增出EGFP和MxA基因的特异性片断,免疫组化结果显示EGFP报告基因在细胞内的阳性表达,并表现出MxA的表达特征,间接证明了MxA可在鸡细胞中表达。MxA基因真核表达载体的成功构建以及在鸡细胞中的表达为进一步研究MxA基因在抗禽病毒性疾病中的应用打下了基础。

Human MxA has broad antiviral spectrum. The aim of the study was to construct eukaryotic expression vector containing antiviral MxA gene which was expressed in chicken cells in order to study the role of MxA against avian virus disease. MxA gene was obtained from pCDNA3.1（＋）-MxA by restriction enzyme EcoRI and ApaI, and linkaged with eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP-C1-MxA was identified by PCR and restriction analysis. pEGFP-C1-MxA was tranfected into chicken embryo fibroblast （CEF） and chicken testicle tissue cells. Granular green fluorescence was detected by fluorescence microscope in cytoplasm of transfected cells after 48 h, and specific fragments of MxA and EGFP gene were amplified respectively by RT-PCR with total RNA of transfected cells. Moreover, the expression of EGFP-MxA fusion protein was detected by immunohistochemistry with monoclonal antibody of GFP. The data indicated that the antiviral gene MxA of the recombinant plasmid could be expressed in CEF and chicken testicle tissue cells. The results would contribute to further studies of application of MxA in dealing with chicken virus diseases.