The Inhibitive Effect Of Diphtheria Toxin A Using Hepatitis B Virus Envelope As Gene Transfer Vector For Hepatocarcinoma Cells

ABSTRACTHepatitis B virus (HBV) has specific tropism of liver. In order to construct an efficientHBV-based vector for targeting the liver, we get hepatitis B virus envelope through themethod ofβ-propiolactone. Diphtheria toxin A chain (DTA) is known to kill cells byribosylating the EF-2 translation-initiation factor and inhibiting protein synthesis. Weconstructed combinant hepatitis B virus envelope (HBVE) vector carrying ps2-DTA tostudy the special targeting and inhibitive effect of the vector for hepatocarcinoma cells invitro.Matirials and Methods1. To get hepatitis B virus envelope, the supernatant fluid of Hep G 2.2.15 wasmixed with a PEGS000 solution for concentration and was inactivated byβ-propiolactone.2. The quality of HBVE was studied with ELISA and electron microscopy. 3. The acquiredhepatitis B virus envelope combined with ps2-DTA was transfected to HepG2,A549 andFB. DTA expression was detected by RT-PCR and immunohistochmistry, cell apopotosiswas detected by TUNEL, and cell survival rate was detected trypan blue resist-dye, and thekill effect was observed by cell morpholoty and biological.ResultsThe acquired hepatitis B virus envelope can retain the surface protein HBsAg+pre S1+pre S2 and can combine with ps2-DTA. Electron microscopy of HBV, HBVE andHBVE=DNA were showed that HBV and HBVE-DNA had electrondense material butHBVE didn't. After ps2-DTA transfected with HBVE, the biological changes of the celland the kill effect on the cell hints: (1).In HepG2 cells, the mRNA and protein came fromDTA increased significantly, and the TUNNEL was increased significantly contrast againstthe control cells. (2).In Hep G 2 cells, cell division phas was seldom, with low survival rateand slow growth. However the control cells proceed into the logarithm growth phase quickly.ConclusionsHBVE, with the Pre S1 retained, can be obtained successfully by the method ofβ-propiolactone. The expression of DTA gene in HepG2 cell carried by HBVE wassignificantly augmented than that carded by liposome and the DTA gene expression inHepG2 cell Then combinant epatitis B virus envelope (HBVE) vector carrying ps2-DTAcan inhibit specically the proliferation of hepatocarcinoma cells.