诺如病毒衣壳蛋白在Tn5细胞中的表达及纯化

Expression of Noro Virus Capsid Protein in Tn5 Cells

摘要:

将人诺如病VA387株ORF2基因（全长1623 bp）插入载体pFastBac1中，转化DH10Bac感受态细胞通过同源重组获得Bacmid-NoV-ORF2；脂质体介导转染sf9昆虫细胞，获得表达NoV-ORF2的重组杆状病毒Ac-NoV-ORF2。Ac-NoV-ORF2感染Tn5细胞，培养5~6 d后，收获细胞；冻融破碎细胞，离心收集上清进行分子筛纯化。SDS-PAGE分析结果显示，重组病毒感染的Tn5细胞可见特异的蛋白条带，接种72 h后衣壳蛋白表达量明显并逐步增加，4 d后产量达到稳定；10% SDS-PAGE分析显示，目的蛋白为相对分子质量59 KD和56 KD的两个条带；电镜观察发现表达的诺如病毒衣壳蛋白成功装配成了大小约为40-50 nm的VLPs。本研究成功的在Tn5细胞中实现了诺如病毒衣壳蛋白的表达和VLPs的装配。

Abstract:

The ORF2 gene sequence of NoVs VA387, at a length of 1 623 bp, was synthesized and inserted into vector pFastBac1. Transformed DH10Bac competent cells by pFastBac1-NoV-ORF which was identified by sequencing to obtain Bacmid-NoV-ORF2. The constructed recombinant plasmid Bacmid-NoV-ORF2 was transfected to sf9 cells by co-precipitation with calcium phosphate. Harvested the supernatant of cells and frozen storage recombinant baculovirus at -20 ℃. Tn5 cells were infected by Ac-NoV-ORF2 virus and the cells was harvested 5 d ~6 d after infection. Break the cells, and the supernatant was harvested and purified by MS purification. Various components were collected, observed by electron microscopy，and determined for protein content by 10% SDS-PAGE. The Tn5 cells transfected with Ac-NoV-ORF2 virus showed specific protein by 10%SDS-PAGE profile. The capsid protein expression was significantly and gradually increase since 3 d after infected, and the production reached stable in 4 d. The 10% SDS-PAGE showed that the capsid protein was mainly located in two bands with relative molecular masses of 59 KD and 56 KD. Electron microscopy showed that the expressed capid protein was assembled to VLPs at sizes of about 40-50 nm. So the capsid protein of NoVs was successfully expressed in Tn5 cells and assembled into VLPs.