鸡SP-A在293T细胞中的表达及免疫学检测

摘要

为表达鸡肺表面活性蛋白A（chicken palmonary surfactant protein A，cSP-A）基因，本研究扩增去除终止子的cSP-A，通过Nhe I和Apa I双酶切，连接进入真核表达载体pcDNA3.1/myc-His(-)A中，构建获得重组载体pcSP-A；同时，将cSP-A和gfp基因按Nhe I-BamH I和BamH I-Apa I分别连接至pcDNA3.1/myc-His(-)A载体中，构建获得融合表达载体pcSP-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cSP-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cSP-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，cSP-A在细胞质中表达，pcSP-A和pcSP-A-GFP融合蛋白的相对分子量分别约为30 kDa和55 kDa。上述研究结果为进一步研究cSP-A的功能奠定了基础。%Chicken pulmonary surfactant protein A (cSP-A) gene was amplifi ed without the stop codon and subcloned into pcDNA3.1/myc-His (-) A vector withNheI andApaI for expression in eukaryotic system. The resulting plasmid pcSPA was subsequently linked with the greenfl uorescence protein (GFP) gene into pcDNA3.1/myc-His (-) A vector with Nhe I/BamH I andBamH I/ApaI sites to construct a recombinant eukaryotic expression vector pcSP-A-GFP. The pcSPA-GFP plasmids were then transfected into 293T cells by TransIT-293 reagent. The sublocalization of cSP-A protein was detected using indirect immunofl uorescence assay (IFA) with the specifi c antibody against cSP-A. The expressed fusion protein was identifi ed in Western blot. Expression ofcSP-A was visualized in cytoplasms of the transfected 293T cells. The relative molecular mass of the fusion proteins pcSP-A-GFP and pcSP-A were about 55 kDa and 30 kDa, respectively. The biological activities of cSP-A expressed in the eukaryotic system will be investigated in the future study.