鸡SP-A在293T细胞中的表达及免疫学检测  

EUKARYOTIC EXPRESSION AND IMMUNOLOGICAL ASSAYS OF CHICKEN PULMONARY SURFACTANT PROTEIN A IN 293T CELLS

作　　者：黄琪[1,2] 汪凯[1,2] 涂健[1] 潘玲[1] 祁克宗[1] 李泽君[2] 陈鸿军[2] 刘红梅[1] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2016年第5期63-68,共6页Chinese Journal of Animal Infectious Diseases

基　　金：安徽省自然科学基金(1408085MC49)

摘　　要：为表达鸡肺表面活性蛋白A(chicken palmonary surfactant protein A,c SP-A)基因,本研究扩增去除终止子的c SP-A,通过Nhe I和Apa I双酶切,连接进入真核表达载体pc DNA3.1/myc-His(-)A中,构建获得重组载体pc SP-A;同时,将c SP-A和gfp基因按Nhe I-Bam H I和Bam H I-Apa I分别连接至pc DNA3.1/myc-His(-)A载体中,构建获得融合表达载体pc SP-A-GFP。通过脂质体法将这两类真核载体瞬时转染293T细胞,48 h后用4%多聚甲醛固定细胞,利用c SP-A特异性鼠多抗进行间接免疫荧光,于倒置荧光显微镜下观察c SP-A表达和亚细胞定位情况,然后收获细胞进行Western blot检测。结果发现,c SP-A在细胞质中表达,pc SP-A和pc SP-A-GFP融合蛋白的相对分子量分别约为30 k Da和55 k Da。上述研究结果为进一步研究c SP-A的功能奠定了基础。Chicken pulmonary surfactant protein A（c SP-A） gene was amplified without the stop codon and subcloned into pc DNA3.1/myc-His（-） A vector with Nhe I and Apa I for expression in eukaryotic system. The resulting plasmid pc SPA was subsequently linked with the green fluorescence protein（GFP） gene into pc DNA3.1/myc-His（-） A vector with Nhe I/Bam H I and Bam H I/Apa I sites to construct a recombinant eukaryotic expression vector pc SP-A-GFP. The pc SPA-GFP plasmids were then transfected into 293 T cells by Trans IT-293 reagent. The sublocalization of c SP-A protein was detected using indirect immunofluorescence assay（IFA） with the specific antibody against c SP-A. The expressed fusion protein was identified in Western blot. Expression of c SP-A was visualized in cytoplasms of the transfected 293 T cells. The relative molecular mass of the fusion proteins pc SP-A-GFP and pc SP-A were about 55 k Da and 30 k Da, respectively. The biological activities of c SP-A expressed in the eukaryotic system will be investigated in the future study.

关 键 词：鸡肺表面活性蛋白A 真核表达 检测 

分 类 号：S852.42[农业科学—基础兽医学]