Establishment Of An Efficient And Marker-Free Recommbination System For Vaccinia Virus Vector

Poxvirus is a kind of large linear double-stranded DNA virus with enveloped genome,including Vaccinia virus,Variola virus,Cowpox virus and Monkey Poxvirus.lt is characterized by multiple non-essential genes,large capacity of exogenous genes(<25kbp)and cytoplasmic replication,so that vaccinia virus and other types of poxviruses can be used as vectors for the construction of vaccines against Human immunodeficiency virus(HIV),Influenza virus,etc.More importantly,vaccinia virus specific gene knockout can make it tumor selective,which can be used as a carrier of oncolytic virus as a new means of tumor immunotherapy.The main methods for obtaining recombinant vaccinia virus vectors include traditional homologous recombination,direct in vitro ligation,bacterial artificial chromosomes,etc.However,these methods have disadvantages such as low recombinant efficiency and long virus purification time,which seriously hinder the clinical application of vaccinia virus vector.Therefore,it is urgent to develop efficient recombinant vaccinia virus vector technology.CRISPR(Clustered regularly interspaced short palindromic repeat)-cas9 is a gene-editing technology in which gRNA guides Cas9 protein to cleave to the target,and has successfully edited the genomes of Adenovirus,Herpes simplex virus,Poxvirus Lister strain and Western Reserve strain.Vaccinia virus Tiantan strain(VTT)is a vaccine strain independently developed in China and has been successfully used to prevent smallpox in the world health organization literature.Therefore.this study intends to establish an efficient reconbinant vaccinia virus vector system based on the vaccinia tiantan strain through CRISPR-Cas9.In this study,firstly,the genome of vaccinia virus was scanned and analyzed,and multiple gRNAs targeted at different sites of vaccinia virus were predicted using the website(http://www.e-crisp.org/E-CRISP/),and TK region was selected for the exploration of efficient recombination technology.Therefore,in this study,after instantaneous transfection of grna-cas9 plasmid or establishment of gRNA-cas9 stable cell line.VTT was infected and transferred into the recombinant plasmid pJ2R-EGFP.After plaque purification and sequencing identification,it was proved that CRISPR-Cas9 technology could edit the TK region of vaccinia virus.Moreover,the recombinant efficiency of this highly efficient recombinant vaccinia virus vector system is much higher than that of the traditional homologous recombinant method,thus establishing a preliminary highly efficient recombinant vaccinia virus vector system.During the establishment of a highly efficient recombinant vaccinia virus vector,exogenous screening marker genes were introduced to facilitate screening.In order to meet the practical application needs of recombinant vaccinia virus,the screening markers in the recombinant virus need to be deleted.Cre-LoxP is a system that uses the Cre enzyme to identify specific DNA sequences(LoxP loci)and make gene recombination(deletion,insertion,translocation)occur between two LoxP loci.Accordingly,the recombinant plasmid with LoxP was constructed In this experiment,and the recombinant virus with LoxP was obtained by efficient recombination method.Then,the screening markers in the recombinant virus were deleted by Cre enzyme,and the recombinant vaccinia virus vector with TK deleted and no screening markers was obtained.To sum up,this study established a highly efficient recombinant and unscreened marker vaccinia virus tiantan strain vector system by using cricrisp-cas9 and Cre-Loxp technology,providing reference value for its vaccine vector construction and tumor immunotherapy.