摘要
目的在293T细胞中表达诺如病毒衣壳蛋白,并进行纯化。方法将合成的人诺如病毒GII.4悉尼大流行株VP1基因（全长1 623 bp）插入真核表达载体pcDNA3.1（＋）中,构建重组表达质粒,测序验证无误后,通过磷酸钙共沉淀法将质粒转染至293T细胞中,收获转染细胞上清,进行Western blot鉴定。转染3~5 d后,收获细胞上清,经氯化铯平衡密度梯度离心纯化衣壳蛋白,收集各组分,离心沉淀病毒样颗粒（virus-like particles,VLPs）,PBS重悬后,SDS-PAGE分析目的蛋白的纯度;电镜观察VLPs;BCA法测定蛋白含量。结果 Western blot分析显示,重组表达质粒转染的293T细胞上清可见特异的蛋白条带。收获的细胞培养上清经氯化铯平衡密度梯度离心后,可见3个明显条带;10%SDS-PAGE分析显示,衣壳蛋白主要位于第3个条带,纯度达95%以上;目的蛋白有2个条带,相对分子质量分别为59 000和56 000;收集的3个组分经电镜观察可见,表达的诺如病毒衣壳蛋白主要位于第3个条带,且组装成了VLPs,颗粒大小约为38 nm;每升培养基可获得约500μg VLPs。结论成功在293T细胞中表达了诺如病毒衣壳蛋白,该衣壳蛋白成功组装成了VLPs。本实验建立了一种小规模制备诺如病毒VLPs的新方法。
Objective To express the capid protein of Norovirus （NoVs） in 293T cells and purify the expressed product. Methods The VP1 gene sequence of NoVs GII. 4 strain causing pandemic in Sydney, at a length of 1 623 bp, was synthesized and inserted into vector pcDNA3. 1 （＋）. The constructed recombinant plasmid was identified by sequencing and transfected to 293T cells by co-precipitation with calcium phosphate. The supernatant of cells was harvested 3 - 5 d after transfeetion, from which capid protein was purified by density gradient centrifugation with cesium chloride, Various components were collected, from which virus-like particles （VLPs） were precipitated by eentrifugation, re-suspended by PBS, analyzed for purity by SDS-PAGE, observed by electron microscopy, and determined for protein content by BCA method. Results The culture supernatant of 293T cells transfeeted with constructed recombinant plasmid showed specific protein bands on Western blot profile. However, the harvested culture supernatant after density gradient eentrifugation with cesium chloride showed three obvious bands. The 10% SDS-PAGE showed that the capsid protein was mainly located in the third band, with a purity of more than 95%. The target protein showed two bands with relative molecular masses of 59 000 and 56 000 respectively. Electron microscopy showed that the expressed capid protein was mainly located in the third band and was assembled to VLPs at sizes of about 30 nm. About 500 μg VLPs were obtained from each liter of medium. Conclusion The capsid protein of NoVs was successfully expressed in 293T cells and assembled into VLPs. A novel method for small scale production of VLPs of NoVs was developed in this paper.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第7期906-909,共4页
Chinese Journal of Biologicals
关键词
诺如病毒
病毒样颗粒
磷酸钙沉淀
Norovirus （NoVs）
Virus-like particles （VLPs）
Calcium phosphate precipitation
