一株表达EGFP基因的重组伪狂犬病毒的构建及其复制稳定性分析

Construction of a Recombinant Pseudorabies Virus Expressing EGFP Gene and Analysis of Its Replication Stability

关键词:  伪狂犬病毒基因缺失株  同源重组  重组病毒  增强型绿色荧光蛋白

Key words:  Gene-deleted pseudorabies virus  homologous recombination  recombinant virus  enhanced green fluorescent protein

摘要:

为探寻伪狂犬病毒（Pseudorabies virus，PRV）基因缺失株rPRV-bc-8基因组UL4-UL3基因间区域是否可作为外源基因的插入位点，在实验室已构建的含有增强型绿色荧光蛋白（Enhance Green fluorescent protein，EGFP）基因的转移质粒pMD-US6+7-EGFP-US2的基础上，通过DNA体外分子克隆技术将UL4序列替换左同源臂US6+7序列，UL3序列替换右同源臂US2序列，并将PolyA引入转移质粒，得到转移质粒pMD-UL4-EGFP-UL3。利用脂质体转染法将转移质粒pMD-UL4-EGFP-UL3与亲本毒rPRV-bc-8共转染PK-15细胞，经蚀斑纯化成功得到一株能够稳定表达EGFP基因的重组伪狂犬病毒rPRV-UL4-EGFP-UL3。通过倒置荧光显微镜观察以及PCR鉴定确定EGFP基因已成功插入到伪狂犬基因组的UL4-UL3之间，并通过细胞传代试验分析得到重组毒的遗传稳定性良好。通过体外增殖试验分析得到重组病毒rPRV-UL4-EGFP-UL3在细胞内的增殖速度较快，接种后12 h内滴度始终高于亲本毒rPRV-bc-8，接种后40 h达到最高滴度10^8.3 TCID₅₀/mL，与亲本毒的最高滴度接近，但是最高滴度的出现时间晚于亲本毒，并且在达到平台期后病毒滴度下降的比亲本毒快。通过对病毒培养液中荧光蛋白浓度进行测定，结果显示在接种后56 h时荧光蛋白浓度达到最高值4793.9 ng/mL，之后进入平台期。综上结果表明构建的重组病毒具有良好的遗传稳定性及体外复制能力，并且外源基因EGFP在此位点具有良好的表达效果，为进一步相关重组病毒的构建奠定了基础。

Abstract:

In order to to explore whether the UL4-UL3 intergenic region in the genome of the gene-deleted pseudorabies virus rPRV-bc-8 can be used as an insertion site for exogenous genes, on the basis of the laboratory constructed transfer plasmid pMD-US6+7-EGFP-US2 of EGFP gene，the left homology arm US6+7 sequence was replaced by the UL4 sequence, and the right homology arm US2 sequence was replaced by the UL3 sequence based on DNA in vitro molecular cloning technology, PolyA was introduced into the transfer plasmid at the same time to get pMD-UL4-EGFP-UL3.The transfer plasmid pMD-UL4-EGFP-UL3 and the parental virus rPRV-bc-8 were co-transfected into PK-15 cells by liposome transfection method. After plaque purification, recombinant pseudorabies virus rPRV-UL4-EGFP-UL3 with stably expressed the EGFP gene was obtained. Through inverted fluorescence microscope observation and PCR identification, it was confirmed that EGFP gene had been successfully inserted into the UL4-UL3 of the pseudorabies genome, and the genetic stability of the recombinant virus was found to be by the analysis of the cell passage test. On the basis of in vitro proliferation test analysis, the recombinant virus rPRV-UL4-EGFP-UL3 proliferates faster in the cells, and the titer within 12 h after inoculation is always higher than that of the parental virus rPRV-bc-8, and reaches the highest titer 10^8.3 TCID₅₀/mL at 40 h after inoculation, which is close to the highest titer of the parent virus, but the highest titer appeared later than the parent virus, and after reaching the plateau, the virus titer decreased faster than the parent virus. By measuring the fluorescent protein concentration in the virus culture medium, the results showed that the concentration of fluorescent protein reached the highest value of 4793.9 ng/mL at 56 h after inoculation，and then entered the plateau phase. The above results show that the constructed recombinant virus has good genetic stability and in vitro replication ability, and the exogenous gene EGFP has a good expression effect at this site, which lays a foundation for the construction of further related recombinant viruses.