SPLUNC1-Ig融合蛋白在CHO细胞中的稳定表达及活性初步分析  

Stable expression and preliminary analysis on biological activity of short palate, lung and nasal epithelium clonel-Igfusionproteininthe CHO cells

作　　者：薛君力[1] 赵雁林 易玲[1] 王小珏[1] 李惠文[1] 韦攀健[1] 郑小芳[1] 张洪涛[1] 

机构地区：[1]北京市结核病胸部肿瘤研究所中心实验室,101149 [2]国家结核病参比实验室

出　　处：《中华生物医学工程杂志》2010年第6期542-547,共6页Chinese Journal of Biomedical Engineering

摘　　要：目的 构建短的上腭、肺及鼻咽上皮克隆1（SPLUNC1）-Ig融合蛋白表达体系并初步分析其活性.方法 在质粒PGEM-T easy-SPLUNC1基础上联合应用大引物法和巢式PCR实现目的序列147位碱基的定点突变,亚克隆于表达载体PDH3,构建PDH3-SPLUNC1的表达质粒.稳定转染CHO/dhfr-细胞完成SPLUNC1-Ig融合蛋白的表达,并在氨甲喋呤（MTX）加压下筛选出稳定表达的细胞株,应用ELISA检测蛋白表达的水平.利用蛋白A亲和层析系统纯化获得的SPLUNC1-Ig融合蛋白,并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹进行鉴定,经ELISA、Western免疫印迹分析SPLUNC1-Ig融合蛋白的特异性和活性.结果 经酶切及测序结果证实预定位点已突变,琼脂糖凝胶电泳出现大小为768 bp的目的条带;测序发现SPLUNC1基因的147位碱基A均已突变成C,证实PDH3-SPLUNC1表达质粒构建成功.RT-PCR扩增出768 bp大小的SPLUNC1基因.ELISA检测CHO/dhfr-细胞上清表明SPLUNC1-Ig融合蛋白在3个月内稳定表达,水平与时间的延长无关.经亲和层析获得SPLUNC1-Ig融合蛋白,SDS-PAGE电泳可见相对分子质量约52 500大小的条带.Western免疫印迹也在45 000与66 200之间出现与预测位置相符的特异目的条带.SPLUNC1-Ig融合蛋白浓度为0.39 mg/L时与脂多糖结合活性明显高于阴性对照,6.25 mg/L时与脂多糖结合的A450值基本达到平台期.Western免疫印迹也显示未变性的脂多糖可与SPLUNC1-Ig融合蛋白结合,而变性后却不能与之结合.结论 建立了非切胶纯化的定点突变方法,顺利实现了表达质粒的构建.成功构建SPLUNC1-Ig融合蛋白表达体系,其分泌的目的蛋白具有蛋白的天然结构和体外活性.Objective To construct expression system in eukaryotic cells the short palate, lung and nasal epithelium clone 1 （SPLUNC1）-Ig fusion protein and to analyze its biological activity. Methods The site-directed mutagenesis of the base 147 in the target sequence was achieved based on PGEM-T easy-SPLUNC1 with megaprimer method and nested PCR. The sequence was then subcloned into the expression vector PDH3 to construct the expression plasmid PDH3-SPLUNC1. Expression of SPLUNC1-Ig fusion protein was achieved in CHO/dhfr- cells stably transfected with PDH3-SPLUNC1. Moreover, cell strains with stable expression of the fusion protein were selected with methotrexate （MTX）, and the expression of protein was determined by enzyme linked immunosorbent assay （ELISA）. The SPLUNC1-Ig fusion protein was purified by protein A affinity chromatography and determined by sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and Western blotting Furthermore, ELISA and Western blotting were used to analyze the specificity and biological activity of the SPLUNC1-Ig fusion protein. Results The mutation was confirmed to be located in the desired site by restriction enzyme digestion and sequence analysis. A target fragment of 768 bp appeared on agarose gel electrophoresis. The sequencing demonstrated A→C mutation at the base 147, which suggested successful construction of the expression plasmid PDH3-SPLUNCI. RT-PCR amplified a SPLUNCI gene with 768 bp in size. ELISA detection of supernatant in CHO/dhfr- cells revealed stable expression of SPLUNC1-Ig fusion protein within three months and the change of expression level was independent of time. SDS-PAGE of the SPLUNC1-Ig fusion protein obtained by affinity chromatography showed a band with relative molecular weight of 52 500. Correlating with this, a specific target band with relative molecular weight between 45 000 and 66 200, in consistency with expected location,appeared in Western blotting. The binding activity of SPLUNC1-Ig fusion protein to lipopolysaccharid

关 键 词：短的上腭 肺及鼻咽上皮克隆1 基因表达 CHO细胞 脂多糖类 免疫活性 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]