Improvement Of Packaging Efficiency Of Lentivirus Vector PSMA-CAR And Its Transfection Into CIK

Objective: 1.To explore the influence of different packaging plasmids and coated plasmids on transfection efficiency in virus packaging.2.To explore the influence of different slow virus packaging methods on transfection efficiency.3.To explore the influence of the total amount of different plasmids and the concentration ratio of transfection reagent on the transfection efficiency.4.To explore the influence of the concentration of transfection enhancer on the transfection efficiency in the process of transfection.Methods: In this experiment,three plasmid lentivirus packaging method was used.1.On the premise of the same packaging method,different packaging plasmids Pdel8.9 and Ps AX2,different coated plasmids VSVG and OG were used for transfection comparison.2.On the premise of the same packaging plasmid:(1)Lip2000 packaging method was used to compare the total amount of transfection reagent and plasmid according to the mass ratio of 1,1.5,2,2.5.(2)PEI packaging method was used to compare the total amount of transfection reagent and plasmid according to the mass ratio of 3,6,9,12.(3)The method of calcium phosphate packaging was used to compare the transfection by adjusting the amount of calcium phosphate in the culture medium;3.In the CIK transfected with CAR,adjust the amount of virus concentrate added in the culture medium and the concentration of Polybrene in the culture medium [mg / ml] to 2,4,6 and 8 respectively for transfection comparison.Result: 1.When PEI was transfected,the plasmids of PSMA-CAR,VSVG and Pdel8.9 were 23.36%,16.32%,22.29% and 32.93% respectively.The plasmids of PSMA-CAR,Ps AX2 and OG were 52.7%,62.47%,64.75% and 63.41% respectively.When the plasmids were PCHD-CAR,VSVG and Pdel8.9,the results were 74.6%,84.3%,84.12% and 56.79% respectively.2.When Lip2000 was transfected,the plasmids of PSMA-CAR,Ps AX2 and OG were 48.78%,57.99%,70.43% and 62.55%,respectively.The results of PCHD-CAR,Ps AX2 and OG were 75.07%,70.17%,74.67% and 83.5%,respectively.3.When using calcium phosphate transfection,The plasmids of PSMA-CAR,Ps AX2 and OG were 5.84%,3.72%,2.36% and 1.51% respectively.4.In the Polybrene group,the results were 3.6%,2.39%,2.20% and 2.38% respectively.Conclusion:1.The transfection efficiency of packing plasmids Ps AX2 and enveloping plasmids OG is higher.2.PEI and Lip2000 had the same transfection efficiency,but Lip2000 was higher.When the ratio of the total amount of plasmids and the amount of Lip2000 added is 1:2,the transfection efficiency is higher.3.The titer of the concentrated virus was more than 5 × 104 TU / ml.4.When PSMA-CAR was transfected with CIK,the concentration of Polybrene in the culture medium was 4 mg / ml,the transfection efficiency was higher.