鸡Mx基因表达及其抗体血清制备的研究-动物遗传育种与繁殖专业论文.docxVIP

II经SDS．PAGE检测，结果显示，在甲醇浓度维持在1％时，鸡抗病毒Mx蛋白在毕 II 经SDS．PAGE检测，结果显示，在甲醇浓度维持在1％时，鸡抗病毒Mx蛋白在毕 赤酵母GSll5中获得良好表达，并且外源基冈表达蛋白约占上清蛋白总量的39．2％。 3．鸡尬基因组成型AAV-293细胞表达系的建立。本研究将鸡胁基因克隆至 plRES2．EGFP载体，并引入6*His标签。鉴定正确后，将质粒plRES2．EGFP．胁 转染AAV-293细胞，荧光倒置显微镜观察外源基因在细胞水平中的表达情况。并 经RT-PCR，Western blotting等方法从RNA、蛋白质水平鉴定该蛋白在细胞中的表 达。结果显示，目的基因实现了在AAV-293细胞中的表达，为稳定表达细胞系的建 立奠定基础。 4．基因免疫制备鸡Mx蛋白抗体血清的研究。采用基因免疫方法，将构建的真 核表达载体pcDNA3．0-彪勉用多聚乙酰亚胺(polyethylenimine，PEI)包裹后经肌肉 途径免疫小鼠，连续免疫4次后，收集抗体血清，以间接免疫荧光(IFA)检测检 测免疫血清抗体的效价，初步制备鸡Mx蛋白抗体血清。IFA结果显示：以1：50 倍比稀释的血清能够特异性识别Mx蛋白；局部放大细胞IFA图片显示Mx蛋白呈 颗粒状分布于细胞质中。 关键词：鸡；磁基因；表达；抗血清 Expression Expression of chicken Agene and preparation of its antibody serum M．S．Candidate：Zhi-quan Tian Advisor：Prof．Bi．chun Li Major：Animal Genetics，Breeding and Reproduction (College ofAnimal Science and Technology，Yangzhou University,Yangzhou 225009) Abstract Mx proteins found in many organisms are type I interferon—induced proteins belonging to dynamic macromolecular enzyme GTP protein family．They have a wide range of antiviral activity and GTP hydrolysis activity．And they Can inhibit negative strand RNA viruses in a wide range．Mx proteins are different in anti—virus specifity and mechanisms．At present，studies about these genes mainly focused on human and mouse， also they had become more and more important in poultry．In this study,chicken Mr gene was subcloned，its prokaryotic expression vector and eukaryotic expression vector were constructed．Then the expression of Mx protein in different vitro conditions and its antibody serum preparation were also achieved．All these above not only laid foundations for the substantial acquisition and detection of chicken Mx protein in vitro，but also provided important references for the selection of disease-resistant genes and the preparation of disease·resistant varieties，transgenic groups in poultry．The major results are as follows： 1．Chicken AZr gene expressed in Escherichia coli and Mx protein Was purified and identified．Its eDNA Was subcloled．and recomb