摘要
猪圆环病毒2型(PCV2)可引起断奶仔猪多系统衰竭综合征,导致仔猪逐渐消瘦。PCV2 ORF1表达的Rep蛋白及其剪切体Rep′是PCV2复制所需的重要蛋白。为了研究PCV2 ORF1部分位点对PCV2复制能力的影响,本试验通过构建PCV2 ORF1区域点突变双拷贝感染性克隆质粒,在无PCV2污染的PK-15细胞中进行病毒拯救,使用荧光定量PCR检测不同位点突变病毒培养不同代次上清液的Ct值。结果:将PCV2 Rep的17 aa、19 aa、20 aa和21 aa突变为丙氨酸后病毒无法成功拯救,2 aa突变后严重影响病毒的复制能力,3 aa、5 aa和18 aa突变后病毒的复制能力增强且细胞病毒载量高于PCV2原毒株,推测17 aa、19 aa、20 aa和21 aa是影响PCV2复制的关键作用位点。本研究结果为未来PCV2复制相关研究提供了试验依据。
Porcine circovirus type 2(PCV2)can cause post-weaning multisystemic wasting syndrome(PMWS),resulting in gradual weight loss in weaned piglets.The Rep protein expressed by PCV2 ORF1 and its splice Repis important for PCV2 replication.In order to determine the effect of the partial sites of PCV2 ORF1 on the replication ability of PCV2,we constructed a point mutation double copy infectious clone plasmid in the PCV 2 ORF1 region and saved it in PK-15 cells without PCV2 contamination.The Ct values of the supernatant of different generations were detected by quantitative PCR after different site mutant viruses were transferred into cells.The results showed that the virus could not be successfully saved after PCV2 Rep 17 aa,19 aa,20 aa and 21 aa sites were mutated into alanine.However,the 2 aa site mutation seriously affected the replication ability of the virus.In addition,the replication ability of the virus was enhanced and the cell loading was higher than that of the original PCV2 strain after the mutation at 3 aa,5 aa and 18 aa sites,respectively.It was speculated that 17 aa,19 aa,20 aa and 21 aa might be the key sites affecting PCV2 replication.This study provided experimental basis for future research on PCV2 replication.
作者
李荷然
肖琦
温立斌
朱雪蛟
芮荣
何孔旺
LI Heran;XIAO Qi;WEN Libin;ZHU Xuejiao;RUI Rong;HE Kongwang(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs/Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis,Nanjing 210014,China)
出处
《畜牧与兽医》
CAS
北大核心
2024年第1期71-76,共6页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金面上项目(31972679)。
