Oncolytic Engineering Of Herpes Simplex Virus Envelope Glycoprotein B Targeting CEA

Background: How to improve the targeting of anti-tumor therapy is the core issue of tumor therapy.Oncolytic virus is a therapeutic virus that has undergone genetic modification to selectively complete the cycle of infection and replication in tumor cells,thereby specifically dissolving and killing tumor cells,and has become a new treatment method for malignant tumors.At present,the development of oncolytic herpes simplex viruses has made great progress.However,there are still many shortcomings that limit the widespread use of oncolytic viruses.For example,the oncolytic virus can only be injected into the tumor to achieve the goal of killing the tumor as much as possible.Modification of g D and g H/g L of herpes simplex virus activates gB,a conserved fusion glycoprotein in the Herpesviridae family,in a cascading manner.gB has been widely used in the study of oncolytic viruses.Carcinoembryonic antigen(CEA)is the membrane protein that plays an important role in tumor occurrence,migration and invasion.CEA is a tumor-specific antigen that is overexpressed in many cancers.CEA has been used in cancer imaging,vaccines,and antibody-based tumor treatments.Based on this,we isolated the herpes simplex virus to modify it,deleted part of the g D(US6)sequence to change the way the virus enters,deleted part of the gB(UL27)sequence,and inserted the single-chain antibody(sc Fv)against CEA into the gB amino acid residues,to detect whether the virus is redirected to CEA-positive cancer cells,to provide new ideas for oncolytic viruses to treat malignant tumors.Purpose: Integrate oHSV with tumor cell markers to construct a new oHSV to maximize its targeted therapy effect,making it more effective and safer to treat tumors.Methods: 1.We have used vero cells for virus isolation and collected virus suspension.We took part of the virus suspension to extract viral DNA,and then sequenced the whole genome of the virus by the second-generation sequencing.2.I used the methods of sequence comparison to compare the sequences of the new strain HSV-1 gB with the sequence of the HSV-1-LXMW gB.3.I determined the gB(UL27)DNA fragment to be modified by searching literature,constructed gRNA vector plasmids using molecular cloning technology,obtained the CEA sc Fv sequence by searching literature and patents,and constructed recombinant plasmids.4.After the molecularly cloned plasmid was transfected,I used the infinite dilution method to select monoclonal cells and verify the transfection efficiency by fluorescence observation and PCR.Results: We cultivated and isolated a Chinese HSV-1 strain.Compared the gB DNA and amino acid sequences of the new strain with the gB DNA and amino acid sequence of our original strain HSV-1-LXMW.The result showed that gB has 14 DNA mutations and two amino acid differences.Four HSV-1 gB gRNA plasmids were successfully constructed,which were targeted to different gB sequences,providing the best gene editing scheme.The infinite dilution method was used to successfully select and verify the monoclonal cells,which laid the foundation for the future construction of oncolytic HSV-1.