CAJ | 학술논문

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.
목적 구건단적상악、폐급비인상피극륭1(SPLUNC1)-Ig융합단백표체체계병초보분석기활성.방법 재질립PGEM-T easy-SPLUNC1기출상연합응용대인물법화소식PCR실현목적서렬147위감기적정점돌변,아극륭우표체재체PDH3,구건PDH3-SPLUNC1적표체질립.은정전염CHO/dhfr-세포완성SPLUNC1-Ig융합단백적표체,병재안갑첩령(MTX)가압하사선출은정표체적세포주,응용ELISA검측단백표체적수평.이용단백A친화층석계통순화획득적SPLUNC1-Ig융합단백,병용십이완기광산납-취병희선알응효전영(SDS-PAGE)화Western면역인적진행감정,경ELISA、Western면역인적분석SPLUNC1-Ig융합단백적특이성화활성.결과 경매절급측서결과증실예정위점이돌변,경지당응효전영출현대소위768 bp적목적조대;측서발현SPLUNC1기인적147위감기A균이돌변성C,증실PDH3-SPLUNC1표체질립구건성공.RT-PCR확증출768 bp대소적SPLUNC1기인.ELISA검측CHO/dhfr-세포상청표명SPLUNC1-Ig융합단백재3개월내은정표체,수평여시간적연장무관.경친화층석획득SPLUNC1-Ig융합단백,SDS-PAGE전영가견상대분자질량약52 500대소적조대.Western면역인적야재45 000여66 200지간출현여예측위치상부적특이목적조대.SPLUNC1-Ig융합단백농도위0.39 mg/L시여지다당결합활성명현고우음성대조,6.25 mg/L시여지다당결합적A450치기본체도평태기.Western면역인적야현시미변성적지다당가여SPLUNC1-Ig융합단백결합,이변성후각불능여지결합.결론 건립료비절효순화적정점돌변방법,순리실현료표체질립적구건.성공구건SPLUNC1-Ig융합단백표체체계,기분비적목적단백구유단백적천연결구화체외활성.
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 lipopolysaccharides (LPS)was significantly higher as compared to the negative control group at a concentration of 0.39 mg/L. A plateau in binding activity to LPS as measured by A450 value was reached when the concentration of SPLUNC1-Ig fusion protein was 6.25 mg/L or over. Western blotting also showed that un- denatured but not denaturedlipopolysaccharide could bind to the SPLUNC1-Ig fusion protein. Conclusions The method of site-directed mutagenesis with non gel-cutting purification is established, which can be used for construction of expression plasmid. A system of SPLUNC1-Ig fusion protein expression is successfully constructed that produces target proteins with natural structure and in vitro activity.