Construction and rescue of infectious cDNA clone of classical swine fever virus based on eukaryotic CMV promoter
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为建立猪瘟病毒(CSFV)反向遗传操作系统,本研究将CSFV石门株(SM)全基因组克隆至pBAC11载体中,并采用重叠延伸PCR方法在病毒基因组5'端和3'端分别加入CMV真核启动子序列和RzBGH序列,并将其基因组nt2809的碱基C突变为T,将原有的Afe I酶切位点突变消失作为拯救病毒的遗传标记.将构建的质粒转染PK-15细胞培养后,收集细胞上清盲传3代后通过RT-PCR扩增蛋白编码基因,并经间接免疫荧光试验(IFA)鉴定.RT-PCR鉴定结果显示扩增到CSFV特异性基因;IFA结果显示接种病毒的细胞出现绿色荧光.表明病毒拯救成功,将其命名为BAC-rSM.将BAC-rSM株在PK-15细胞中连续传18代,将第18代病毒经RT-PCR扩增E2基因,并对遗传标记的位点经AfeI酶切和鉴定,分析BAC-rSM病毒的遗传稳定性,结果显示第18代BAC-rSM株遗传标记稳定存在,表明拯救的病毒具有遗传稳定性.将BAC-rSM和SM株病毒分别以MOI 1感染PK-15细胞,在感染后不同时间点收获病毒并测定病毒滴度,结果显示BAC-rSM和SM株生长动力学特征基本一致.对第18代BAC-rSM株与SM株进行全基因组序列比对分析,利用在线软件npsa-prabi.ibcp.fr/对Erns和E2蛋白二级结构预测,采用荧光定量PCR(qPCR)对BAC-rSM和SM株感染PK-15细胞中IFN-β mRNA的转录水平.全基因测序比对分析结果显示,BAC-rSM株基因组共存在46个碱基突变,经预测其Erns和E2的突变导致各自的二级结构改变;qPCR检测结果显示,相对SM感染的PK-15细胞,BAC-rSM感染后8 h和24 h PK-15细胞中IFN-β mRNA的转录水平显著或极显著降低(P＜0.05、P＜0.01).本研究建立了一种简单、操作方便的CSFV反向遗传操作系统的方法,为进一步开展CSFV的分子生物学、毒力决定因素、分子发病机制、疫苗开发和病毒与宿主相互作用的研究提供了新的技术方法.
In order to establish the reverse genetics system of classical swine fever virus(CSFV),the whole genome of CSFV Shimen strain was cloned into pBAC11 vector,and the CMV eukaryotic promoter sequence,RzBGH sequence were added to the 5'end and 3'end of the viral genome by overlap PCR.A genetic marker was generated by the mutation C to T at 2809 of the genome,resulting in the disappearance of the original Afe I restriction site on the rescued virus.The constructed plasmid was then transfected into PK-15 cells and the supernatant was collected and blindly passed for three times.The rescued virus was identified by RT-PCR and indirect immunofluorescence assay(IFA).The specific gene of CSFV was amplified by RT-PCR,and the IFA results showed green fluorescence,indicating that the virus was successfully rescued and named BAC-rSM.The BAC-rSM strain was continuously passed into PK-15 cells for 18 generations.The E2 gene of the 18th generation virus was amplified by RT-PCR and the Afe I sites containing genetic markers were identified by restriction endonuclease digestion and sequencing to identify the genetic stability of BAC-rSM virus.The whole genome of BAC-rSM was sequenced and compared with its parent virus SM strain.The results showed that the genetic markers of the 18th generation of BAC-rSM were stable,indicating that the virus had genetic stability.PK-15 cells were infected with BAC-rSM and SM virus with MOI 1,and the virus was harvested at 4 hours,8 hours,12 hours,24 hours,36 hours,48 hours,72 hours and 84 hours post-infection,and the virus titer was determined.The results showed that the growth kinetics of BAC-rSM and SM were basically the same.The whole genome sequencing analysis of BAC-rSM was performed and compared with the parental virus SM strain.The secondary structure of Erns and E2 protein was predicted by online software npsa-prabi.ibcp.fr/,and the IFN-β of PK-15 cells infected by BAC-rSM and SM strains was quantitatively analyzed by qPCR.The whole gene sequencing analysis of the rescue virus BAC-rSM showed that there were 46 base mutations in the genome,and the secondary structure changes caused by mutations in Erns and E2 were predicted.The relative quantitative detection of IFN-β induced by SM and BAC-rSM infection in vitro showed that there was a significant difference between 8 hours and 24 hours compared to cells infected with SM strain.In this study,a simple and convenient CSFV reverse genetic operating system was established,which provides a new technical approach for advancing research in various areas of CSFV biology,including identification of virulence determinants,understanding molecular pathogenesis,vaccine develop-ment and exploring virus-host interaction.
classical swine fever virusCMV promoterinfectious cloningreverse genetic operating system
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万方数据
