Construction And Preliminary Application Of The CRISPR/Cas9 Knockout Vector Library Of African Swine Fever Virus

African swine fever(ASF)is a highly contagious disease caused by African swine fever virus(ASFV).China has classified it as a type of disease,and the World Organisation for Animal Health(OIE)has identified it as an animal epidemic that must be reported.The CRISPR/Cas9 system was originally discovered as a natural microbial immune mechanism to prevent invasion of viruses or other genetic components.The adaptability of this approach is to achieve efficient genome editing using site-specific DNA double-strand breaks,allowing the native CRISPR system to specifically edit the mammalian genome with very high accuracy.Researchers have done a lot of work on immune adjuvants,attenuated vaccines,DNA vaccines,and subunit vaccines(p72,p30,p54,p12),but unfortunately,clinical trials have shown that these vaccines do not provide good protection effectiveness.Recent studies have found that the establishment of the African CSFV CRISPR/Cas9 knockout technology platform will provide the possibility to develop an effective live vaccine for African swine fever gene deletion.In this paper,the following content studies were carried out on the construction and preliminary application of the HIV/Cas9 knockout vector library of African swine fever virus:1.The CD2v and UK(DP96R)promoters of ASFV SY18 strain were cloned,and recombinant vectors containing different promoters and eGFP reporter genes were constructed respectively.The transfectants were transfected into human embryonic kidney cells(293T),porcine kidney cells(PK15),African green monkey kidney cells(Vero)and passaged porcine alveolar macrophages(IPAM)to observe promoter activity,and further studied ASFV SY18 strain.The same region starts characteristics and mechanisms of child-specific transcriptional regulation of mammalian cells.2.Synthesis of resistin-synthesizing peptides and antibodies thereof by synthesizing five KLH-conjugated polypeptides against CD2v protein.It provides a useful tool for the study of the molecular function of ASFV CD2v protein and the mechanism of CD2v protein.3.Construct and screen recombinant gRNA(guide RNA)expression plasmid based on 49 genes of African swine fever,establish ASFV genome CRISPR knockout gRNA library,and identify the cleavage efficiency and immunological activity against the gRNA of P30 gene for subsequent follow-up The genes laid the foundation for the development of appropriate immune control strategies for African swine fever.A rapid and accurate molecular detection method for African swine fever virus against SY18 strain P72 gene was established,which provided a necessary molecular diagnostic tool for the rapid diagnosis of swine fever in Africa.