Molecular Identification of Self-excision of Bacterial Artificial Chromosome Sequence in Meq-null Marek's Disease Virus

The test was aimed to study the molecular identification of self-excision of bacterial artificial chromosome in meq-null Mareku0027s disease virus(MDV)clone genome.SC9-1recombinant plasmid containing cre recombinase expression cassette was transfected into chickens embryo fibroblast(CEF)to rescue SC9-1recombinant virus.SC9-1recombinant virus was passaged on CEF cells continuously,using cre recombinase knocked out BAC sequence in process of virus passaged.Extracting 1to 10 generations DNA of SC9-1recombinant virus as template,PCR detected BAC sequence of viral genome by specific primers.Using specific primers for the BAC sequence of gpt gene,the MDV conserved gene pp38 as an internal standard,the content of BAC sequences in theviral genome of different passages were detected by fluorescence quantitative PCR.The residual sequence of both sides of loxp site were amplified by PCR,and future verifying the knockout of BAC sequence by sequencing.The results showed that,using the BAC sequence specific primers to detect BAC sequence of different generations in the viral genome,the first five generation of virus DNA could amplify 600 bp specific band meaning the viral genome contained the BAC sequence.The 6to 10 generationes of virus DNA couldnu0027t amplify 600 bp specific band meaning the viral genome didnu0027t contain the BAC sequence.The result of fluorescence quantitative PCR showed that the content of BAC sequence in viral genome decreased gradually with the passage of the virus and couldnu0027t detect BAC sequence completely until the sixth generation.In the process of the knockout of BAC sequence,PCR identifying the residual sequence of both sides of loxp site,the results showed only one loxp site left after the knockout of BAC sequence and the sequence homology was above 99.7%.The experiment confirmed that the cre/loxp system could delete the BAC sequence in MDV meq-null mutant SC9-1completely by culturing virus on CEF cells for 6generation of batches and the knockout of BAC sequence had high degree of consistency.