To construct an efficient and easily manipulated reverse genetics platform for the infectious bronchitis virus(IBV)genome,we employed transformation-associated recombination(TAR)technology to rapidly construct infectious clones in Saccharomyces cerevisiae.Firstly,the genome of IBV H120 strain was amplified using RT-PCR for 9 fragments,and the human cytomegalovirus(CMV)promoter sequence was introduced at the 5'end of the genome,while the hepatitis D virus ribozyme sequence(HDVRz)and bovine growth hormone(BGH)polyadenylation signal sequence(subsequently called HB sequence)were introduced at the 3'end.A silent mutation,C20356T,was introduced into fragment F7 by primer design as a genetic marker.Subsequently,full-length cDNA clones of the H120 genome were rapidly constructed by transforming 9 DNA fragments and linear plasmid pYES1L into Saccharomyces cerevisiae cells using the TAR cloning technique.The recombinant plasmid pYES1L-H120 was obtained by Junction-PCR screening and sequencing.To improve the efficiency of virus rescue,a helper plasmid expressing the N gene of IBV,pcDNA3.1-N+3'UTR,was also constructed.BHK-21 cells were co-transfected with pYES1L-H120 and the helper plasmid pcDNA3.1-N+3'UTR.The cells were harvested 48 hours post transfection,then inoculated into 9-day-old SPF chicken embryos and passed through 3 generations.The results of somatic lesions and sequencing showed that the rH120 strain of IBV reverse genetic virus was successfully obtained.Additionally,in order to express exogenous genes using IBV as vector,we used the TAR cloning technique to construct a cDNA clone expressing the exogenous eGFP gene by replacing the 5a gene of the viral genome with the eGFP gene,and the chimeric virus rH120-Δ5a-eGFP was successfully rescued.The results showed that IBV reverse genetic recombination virus could be obtained in a short time by using TAR cloning technique.The viral genome could also be manipulated quickly to obtain the chimeric virus with stable expression of exogenous genes while using IBV as the vector.In this study,the IBV reverse genetic operating platform based on TAR cloning technology was successfully established for the first time,which platform provides an efficient and convenient tool for studying the biological characteristics of viruses,the development of vector vaccines and the screening of antiviral drugs.
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