摘要
为构建表达Strep-tag II的重组塞内卡病毒(SVA),本研究在感染性cDNA克隆p SVA-I212V/S460L的基础上,采用重叠延伸PCR扩增Strep-tag II并采用无缝克隆法将其插入SVA VP1基因C端,构建感染性cDNA克隆,命名为p SVA-Strep,并采用双酶切和测序鉴定正确后,将p SVA-Strep转染BHK-21细胞拯救重组病毒,收集病变细胞上清,并将含有病毒的细胞上清和野生型SVA(SVA-WT)分别感染BHK-21细胞,以未感染病毒的BHK-21细胞作为阴性对照,感染后24 h分别以鼠源SVA VP2蛋白单克隆抗体(MAb)5D10(1∶1000)、鼠源Strep-tag II MAb(1∶2000)作为一抗,以FITC标记的山羊抗小鼠Ig G(1∶2000)为二抗,采用间接免疫荧光试验(IFA)对重组病毒鉴定。IFA结果显示,以鼠源SVA VP2蛋白MAb 5D10为一抗,SVA-WT和重组病毒感染的细胞均出现绿色荧光,以Strep-tag II MAb作为一抗时,仅有重组病毒感染的细胞出现绿色荧光,经SVA-WT感染的BHK-21细胞及阴性对照细胞均无绿色荧光,表明拯救了Strep-tag II标记的重组病毒,并将其命名为r SVA-Strep。将r SVA-Strep在BHK-21细胞中连续传10代,每隔2代采用引物VP1-F/R经PCR鉴定并对第10代重组病毒的VP1基因测序以鉴定r SVA-Strep的遗传稳定性。结果显示,每隔两代的重组病毒经PCR扩增后均出现约780 bp的目的条带,第10代重组病毒扩增的VP1基因测序结果显示Strep-tag II基因仍在重组病毒中,且无基因突变,表明r SVA-Strep的遗传稳定性较强;将SVA-WT和r SVA-Strep分别以MOI 0.01感染BHK-21细胞,在感染后4 h、8 h、12 h、24 h、36 h、48 h和72 h收获病毒测定病毒滴度并绘制生长曲线,结果显示在病毒感染36 h内r SVA-Strep和SVA-WT生长动力学基本一致,表明Strep-tag II的引入在病毒感染36 h内对SVA的复制基本无影响。采用0.2%甲醛充分灭活r SVA-Strep,并利用Strep-TactinRXT Purification纯化试剂盒纯化该灭活病毒,将r SVA-Strep灭活病毒上清加至Strep-Tactin亲和层析柱,分别用不同体�
In order to construct Senecavirus A expressing Strep-tag II,Strep-tag II was inserted into the C-terminus of the VP1gene of SVA-I212V/S460L infective clone by overlapping extended PCR method.The reconstructed infectious clone was then transfected into BHK-21 cells to rescue the recombinant virus,and the supernatant was collected after CPE was detected.BHK-21cells were infected with the virus supernatant and wild type SVA,respectively,and the BHK-21 cells without virus infection were used as negative control.Cells infected with recombinant virus were identified 24 hours after infection by indirect immunofluorescence assas(IFA),mouse anti-SVA VP2 protein MAb 5D10 and mouse anti-Strep-tag II MAb were used as primary antibodies,respectively.IFA results exhibited that both SVA-WT and r SVA-Strep infected cells showed specific fluorescence signals when mouse anti-SVA VP2 protein MAb 5D10 was used as the primary antibody,while only recombinant virus infected cells showed specific fluorescence signals when anti-Strep-tag II MAb was used as the primary antibody.None of the negative controls showed a special fluorescent signal,indicating that the Strep-tag II-labeled recombinant virus was successfully rescued and named r SVA-Strep.To determine the genetic stability of r SVA-Strep,the recombinant virus was passaged in BHK-21 cells for 10 continuously generations.The recombinant virus gene was amplified with primer VP1-F/VP1-R every 2 generations,and the amplified gene of the 10th generation was sequenced.The result displayed that the target bands of about 780bp were present in all the amplified recombinant virus genes,and no mutantion was detected in Strep-tag II gene of the 10th generation,indicating that Strep-tag II gene was stable in r SVA-Strep replication.BHK-21 cells were infected with either SVA-WT or r SVA-Strep with a multiplicity of infection(MOI)of 0.01,and the virus were harvested at 4 hours,8 hours,12 hours,24 hours,36 hours,48 hours and 72 hours after infection and the virus titer was determined.The results s
作者
史家宝
蒙靓
肖培宇
范峻豪
安同庆
王海伟
于力
SHI Jia-bao;MENG Liang;XIAO Pei-yu;FAN Jun-hao;AN Tong-qing;WANG Hai-wei;YU Li(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第2期140-146,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省重点研发计划项目(JD22A023)。
