北京地区人偏肺病毒核蛋白基因的原核表达及抗原活性分析

Prokaryotic expression and antigenicity of nucleocapsid protein gene from human metapneumovirus in Beijing

目的用大肠杆菌表达获得人偏肺病毒主要结构蛋白N蛋白,为下一步的深入研究奠定基础。方法从重组质粒pUCm-N1816中PCR扩增获得N基因,用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET30a(+)中,得到重组表达质粒pET30a-N1816,转化大肠杆菌BL21(DE3),IPTG诱导培养。SDS-PAGE和Westernblot检测目的蛋白的表达和抗原性。结果经双酶切和测序证明1.2kbN1816基因正确插入pET30a中,并具有正确的读码框架,得到预期的pET30a-N1816重组原核表达质粒。37℃,1mmol/LIPTG诱导培养6h后产生大量带6个组氨酸标记的重组N蛋白,主要以包涵体形式存在,占细胞总蛋白的20%左右。N蛋白粗提物经Co2+亲和层析获得较理想纯化。Westernblot结果显示,体外所表达的蛋白能和特异性抗血清及人血清反应。结论本研究成功构建了重组pET30a-N1816原核表达质粒,N蛋白获得了高效表达,并且具有特异抗原活性,可用于人偏肺病毒的深入研究。

Objective To express the nucleocapsid protein（N） gene of human metapneumovirus（hMPV） recently identified in Beijing in E. coli. Methods N gene was amplified from recombinant plasmid pUCm-N1816 by PCR with primers and then sub-cloned into the pE330a（ ＋ ） vector, a prokaryotic expression vector, after dualenzyme digestion with BamH Ⅰ and EcoR Ⅰ . The pE330a-N1816 was transformed into E. coli BL21（DE3） and expressed. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. Results The recombinant plasmid pE330a-N1816 has correct open reading frame confirmed by dual-enzyme digestion analysis and sequencing. The fusion protein 6·His-N was produced and with 20% expression level in terms of total bacterial protein after inducing by 1 mmol/L IPTG at 37℃ for 6 h. A unique protein band of approximate 45 kD was characterized by SDS-PAGE dectrophoresis and purified by Co^2＋ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against pelypeptides of the nucleoeapsid protein of hMPV and to human sera. Conclusion The recombinant plasmid pET30a-N1816 was successfully constructed. The N gene was highly expressed in prokaryotic system and the expressed N protein has specific antigenic activities. It can be used for the further studying of hMPV infections in Beijing.