Construction And Packaging Of Adenovirus Vectors Containing ?1,3GT And E1A

At present,all kinds of malignant tumors are still the main diseases that affect human health and even life threatening.According to research data by 2018,there were about 18.1 million new cancer cases and 9.6 million cancer deaths worldwide.The incidence and mortality rate of cancer in China remain high,among which lung cancer,stomach cancer,colorectal cancer,liver cancer and breast cancer are still among the top five in the world.Although traditional tumor treatment methods such as surgery,radiotherapy and chemotherapy can achieve good results in a short time,the toxic and side effects and residual components of tumor cells often lead to an increase in metastasis and recurrence rate of tumor cells.In addition,it can cause damage to the body itself,especially the hematopoietic system and immune system.Therefore,traditional tumor therapy is difficult to achieve better long-term effect for patients with tumor metastasis.However,with the further development of molecular mechanism of tumor,gene therapy for cancer through gene transfer technology has been widely applied.Currently,there are mainly 2 carriers for tumor gene therapy.One is viral vector.The other is non viral vectors.Because viruses are more likely to invade human cells and affect the transcription and translation of host genes,viral vectors are more widely used than non viral vectors.Adenovirus vectors carrying foreign gene ?1,3 galactosyltransferase can synthesize ?-Gal epitopes on the surface of tumor cell membranes,thereby amplifying the immunogenicity of tumor associated antigen in tumor cells.Specifically,it stimulates cellular immunity and humoral immunity.It can produce stable and efficient CD4/CD8 T cell responses by inhibiting the antigen presenting lymphoid tissue,inhibiting tumor growth and further killing tumor effect.The Early region 1A gene is a tumor suppressor gene that regulates the expression of multiple genes and induces programmed cell death through dependent or non p53 dependent pathways.Regulating cell cycle and keeping cells in G1 and G2 phases.It is found that Early region 1A has no carcinogenic effect on human cells.In fact,Early region 1A acts as a tumor suppressor,that is,anticancer gene.The inhibitory effect of Early region1 A may be related to the regulation of many genes.Experiments have proved that it can inhibit tumor growth,invasion and metastasis in many ways.At the same time,the killing effect of NK cells,cytotoxic T lymphocytes and macrophages has been improved.The clinical application of Early region 1A gene has been approved by FDA and has achieved positive results through clinical trials.Therefore,the clinical application of Early region 1A gene is of great significance to improving the quality of life and improving the survival rate of patients with cancer.Human telomerase reverse transcriptase is positive in 85% of human tumor tissues.Therefore,the h TERT promoter can specifically drive the expression of therapeutic genes in tumor cells,thereby achieving the targeting of tumor gene therapy and reducing the toxic and side effects of gene therapy on normal tissues.Therefore,the h TERT promoter can be used as an ideal promoter for tumor targeted therapy genes to initiate certain genes,such as Early region 1A,to replicate and express in tumor cells.The recombinant adenovirus vector with high expression of foreign genes,?1,3 galactosyltransferase and Early region 1A,was constructed in order to package oncolytic adenovirus with the ability of killing tumor,so as to provide a basis for exploring effective tumor targeting therapy drugs.Objective: To construct three recombinant adenovirus vectors p Ad-h TERT-?1,3GT-E1A-EGFP,p Ad-h TERT-?1,3GT-EGFP and p Ad-h TERT-E1A-EGFP by Gateway technology,and to package recombinant adenovirus Ad-h TERT-?1,3GTE1A-EGFP,Ad-h TERT-?1,3GT-EGFP,and Ad-h TERT-E1A-EGFP by liposome transfection.Methods: In this study,primers were designed to amplify the target genes for PCR amplification,and then to create an entry clone,that is,cloned the target gene h TERT-?1,3GT-EGFP,E1 A and h TERT-E1A-EGFP into the p Entry by homologous recombination reaction,thus constructing the entry clone p Entryh TERT-?1,3GT-E1A-EGFP,p Entry-h TERT-?1,3GT-EGFP,and p Entry-h TERT-E1A-EGFP.Then constructing the expression vector p Ad-h TERT-?1,3GT-E1 AEGFP,p Ad-h TERT-?1,3GT-EGFP,p Ad-h TERT-E1A-EGFP by mixing the entry clone containing the target gene and the target vector p Ad and Gateway LR Clonase enzyme.Recombinant adenovirus Ad-h TERT-?1,3GT-E1A-EGFP,Ad-h TERT-?1,3GT-EGFP,Ad-h TERT-E1A-EGFP was transfected into HEK293 cells by liposome transfection.Reverse transcription polymerase chain reaction was used to identify the virus.The recombinant adenovirus was infected in different tumor cells,and the replication ability of the recombinant adenovirus in tumor cells was verified by indirect immunofluorescence of the corresponding antibodies.The inhibitory effect of the recombinant adenovirus on tumor cells was detected by CCK-8.Results: The target gene was constructed by PCR with specific primers.The primer clone was constructed by homologous recombination.The recombinant adenovirus vector was constructed by LR reaction.The recombinant adenovirus was packaged by liposome transfection.The indirect immunofluorescence assay showed that the recombinant adenovirus could be replicated in tumor cells in large quantities.The recombinant adenovirus Ad-h TERT-?1,3GT-E1A-EGFP was demonstrated by CCK-8 in vitro,this indicated the inhibitory effect of recombinant adenovirus Adh TERT-?1,3GT-E1A-EGFP on human tumor cells.Conclusion: The recombinant adenovirus Ad-h TERT-?1,3GT-E1A-EGFP constructed and packaged in this study is a double specific recombinant adenovirus with tumor specific killing and tumor specific replication ability.