Study On The Therapeutic Effect Of Adeno-Associated Virus Of Recombinant ENO1 Monoclonal Antibody Variable Region Gene On Cervical Cancer Cells

Objective We developed an adeno-associated virus（rAAV-ENO1mAb-5）with the variable region gene of recombinant ENO1 monoclonal antibody,which was mediated to target the variable region gene of recombinant ENO1 monoclonal antibody into the cytoplasm to study the therapeutic effect of rAAV-ENO1mAb-5 on the growth of cervical cancer Hela cells in vitro.Method 1.The variable region sequence of ENO1mAb-5 was selected on the basis of the previous laboratory,and Linker--（GGGGS）₃was used in the middle,Xba I and Hind III were used as digestion sites,and cloned into pAAV2-MCS vector to obtain recombinant plasmid AAV-ENO1mAb-5.The recombinant plasmid AAV-ENO1mAb-5 and helper plasmid（p Helper and pAAV-RC）were co-transfected into AAV293 cells to obtain rAAV-ENO1mAb-5.Meanwhile,pAAV-IRES-Zs Green5with green fluorescent protein was transfected into AAV293 cells as an empty vector control（rAAV-GFP）.2.Na Cl-PEG8000 and glacial ethanol precipitation were used for crude purification and concentration,and the titers of the two were determined by TCID50 method.3.The inhibitory effect of rAAV-ENO1mAb-5 on the glycolytic products（pyruvate and lactic acid）of Hela cells was determined by the glycolysis kit.4.The inhibitory effect of rAAV-ENO1mAb-5 on the growth of cervical cancer Hela cells was detected in vitro by MTT test and scratch test.Results 1.rAAV-ENO1mAb-5 and rAAV-GFP were successfully prepared with a transfection efficiency of 90%and titers of 2.0×10⁷PFU/ml.2.rAAV-ENO1mAb-5significantly inhibited the production of glycolytic products（pyruvate and lactic acid）in Hela cells（P<0.01）.3.When the MOI value of rAAV-ENO1mAb-5 reached 80,the proliferation rate of Hela cells was（48.38±3.94）%,which was statistically significant compared with the rAAV-GFP group and PBS group（P<0.001）.4.In the scratch test,rAAV-ENO1mAb-5 had a significant migration inhibition effect on Hela cells,with statistically significant difference compared with the rAAV-GFP group and PBS group（P<0.01）.Colusion 1.The variable region gene of rAAV-ENO1mAb-5 was successfully transferred into the cytoplasm with high efficiency a nd targeted loading of ENO1monoclonal antibody;2.rAAV-ENO1mAb-5 can significantly inhibit the production of lactic acid and pyruvate in cervical cancer Hela cells;3.rAAV-ENO1mAb-5 can inhibit the proliferation and migration of cervical cancer cells.