Construction Of Recombinant Oncolytic Adenovirus Carrying Melittin Gene And Study On Their Specific Inhibition To The Proliferation Of Hepatoma Carcinoma Cells

OBJECTIVE: To construct an oncolytic adenovirus, which the hybrid promoter HREAFP regulates the E1a, and E1b with 55 kda deleted, carrying Melittin gene which is under the control of the major late promoter and to observe the specific infection and replication capability of the adenoviruses in different cell lines and the specific inhibition to the proliferation effect of QG511-HA-Melittin to different cell lines.METHODS: We performed a LR recombination reaction between the plasmid pENTR-MLP-Melittin and the plasmid pPE3-F11B-MLP-Melittin to generate the new plasmid pPE3-F11B-MLP-Melittin. To construct the new desired recombinant adenoviruses QG511-HA-Melittin, the plasmid PE3-F11B-MLP-Melittin, with the melittin gene, and the plasmid PXC20-△E1b-55kda-HREAFP were co-transfected into 293 cells by homologous recombination, mediated by Lipofectamine 2000 .Then the recombinant adenoviruses were confirmed by PCR analysis using specific primers, amplified at a large scale and purified by cesium chloride gradient centrifugation. The viral titer of QG511-HA-Melittin was tested with TCID50 method. For the purpose of comparing the infection and replication capability of the adenoviruses, viral replication experiments were performed to evaluate the selective replication ability of QG511-HA-Melittin in different cells. The tested tumor cell lines and normal cells are the AFP-positive tumor cell lines HepG2, Hep3B, the AFP-negative tumor cell lines SMMC-7721, and the normal cells LO2. MTT experiments were carried out to observe the specific inhibition to the proliferation effect of QG511-HA-Melittin to different cell lines. The tested tumor cell lines and normal cells are the AFP-positive tumor cell linesHep3B, the AFP-negative tumor cell lines SMMC-7721, and the normal cellsLO2.RESULTS: Recombinant oncolytic adenovirus containing melittin gene were successfully constructed, the recombinant adenoviruses were confirmed by PCR analysis using specific primers. We got the corresponding straps,HRE166bp,AFP276bp,△E1b-55kda1832bp,MLP-Melittin534bp. Viral replication experiments indicated that the Viral replication times of QG511-HA-Melittin in Hep3B cells at 48 hour was 12800, and 130 times in SMMC-7721, and 19.68 times in L02 cells. MTT experiment manifested that the replication of QG511-HA-Melittin was remarkable in AFP-positive cells such as Hep3B ,it was MOI=5 that QG511-HA-Melittin could kill half of the Hep3B cells, while hardly replicated in AFP-negative cells SMMC-7721 and normal cells L02. Compared with the SMMC-7721 and L02 cells, it demonstrated significant cells growth inhibition (P<0.05)CONCLUSIONS: Recombinant oncolytic adenovirus containing melittin gene were successfully constructed, and melittin gene which was regulated by MLP promoter, was successfully inserted into the recombinant oncolytic adenovirus. The in vitro experiments manifested that the replication of QG511-HA-Melittin was remarkable in AFP-positive cells, while hardly replicated in AFP-negative cells and normal cells, and QG511–HA -Melittin could indeed specifically inhibit proliferation of AFP-positive cells. In conclusion the virus exerts the potent anti-tumor efficacy and low side toxicity. It provided a new strategy for liver cancer biotherapy.