猫杯状病毒反向遗传系统的建立及弱毒株的构建  

Construction and application of reverse genetic system for feline calicivirus

作　　者：于雯 孙慧敏 方晨捷 宋家升 鲍恩东[1] YU Wen;SUN Huimin;FANG Chenjie;SONG Jiasheng;BAO Endong(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Zhejiang Difference Biotechnology Co.,Ltd.,Hangzhou 310000,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]浙江迪福润丝生物科技有限公司,浙江杭州310000

出　　处：《畜牧与兽医》2024年第5期107-112,共6页Animal Husbandry & Veterinary Medicine

基　　金：江苏省农业科技自主创新资金项目[CX(22)2016];南京农业大学校企合作项目(技术服务类)。

摘　　要：为了构建猫杯状病毒(FCV)弱毒疫苗候选株,从杭州流行的FCV毒株FCV-HZ-DF-19中提取基因组RNA,利用RT-PCR技术分3段扩增获得FCV全基因组,通过同源重组的方法将全长基因组克隆至已插入榔头状核酶(HamRz)和丁型肝炎核酶(HdvRz)的PCI载体,获得中间质粒pPCI-FCV;以pPCI-FCV为模板扩增获得含HamRz、HdvRz和FCV全基因组的产物FCV-H,并将其连接到BAC载体中获得含有FCV全基因组的克隆pBAC-FCV。将pBAC-FCV转染猫肾细胞F81连续传代后收获病毒。针对FCV毒力相关的LC基因进行缺失,获得感染性克隆pBAC-FCV-ΔLC,通过转染表达FCV-VP1蛋白的猫肾细胞系进行病毒拯救。结果显示,拯救毒株rBAC-FCV和LC基因缺失毒株rBAC-FCV-ΔLC均可产生细胞病变;测序结果表明,拯救毒株的基因序列与亲本毒株FCV-HZ-DF-19一致,且生长曲线与亲本毒相似,而基因缺失毒株的生长则慢于亲本毒株,产生的噬斑大小也明显小于亲本毒株,表明缺失LC基因后病毒毒力下降。结论:本研究成功构建了FCV的反向遗传系统及基因缺失弱毒株,为后续弱毒疫苗研发和FCV生物学特性研究奠定了基础。In order to construct a candidate strain for attenuated feline calicivirus(FCV)vaccine,genomic RNA was extracted from the prevalent FCV strain FCV-HZ-DF-19 in Hangzhou.The entire FCV genome was amplified in three segments using RT-PCR technology.The full-length genome was cloned into a PCI vector containing hammerhead ribozyme(HamRz)and hepatitis D ribozyme(HdvRz)through homologous recombination,and the intermediate plasmid pPCI-FCV was obtained.Using pPCI-FCV as a template,the product FCV-H con-taining the entire genome of HamRz,HdvRz,and FCV was amplified,and connected to a BAC vector to obtain a cloned pBAC-FCV contai-ning the entire genome of FCV.Then,pBAC-FCV was transfected into cat kidney cell F81 for continuous passage in order to harvest the vi-rus.Deletion of LC genes related to FCV virulence,acquisition of infectious clone pBAC-FCV-ΔLC,and virus rescue were performed by transfecting cat kidney cell lines expressing the FCV-VP1 protein.The results showed that the rescued strain rBAC-FCV and the LC gene deficient strain rBAC-FCV-ΔLC could both cause cellular lesions.The sequencing results showed that the gene sequence of the rescued strain was indeed consistent with the parent strain FCV-HZ-DF-19,and its growth curve was similar to that of the parent strain.However,the growth of the gene deficient strain was slower than that of the parent strain,and the size of the generated plaque was significantly smaller than that of the parent strain,which indicated a decrease in virus virulence after the LC gene was deleted.The above results suggested that the reverse genetic system of FCV was successfully constructed in this study,and a gene deficient attenuated strain was constructed;which laid the foundation for the development of attenuated vaccines and the study of the biological characteristics of FCV in the future.

关 键 词：猫杯状病毒 反向遗传学技术 同源重组 基因缺失 

分 类 号：S852[农业科学—基础兽医学]