Study Ofantitumor Effect Of Recombinant Adenovirus Vector Ad-TRAIL-2A-HN On Marek’s Disease Oncogenesis MDCC-MSB-1

Marek’s Disease (MD) is a lymphocyte proliferative disease of chicken whichcaused by Marek’s Disease Virus (MDV).As the only animal viral oncosis which canbe prevented and control effectively by vaccine application so far, MD has greatrefence value to prevention and control of cancer of poultry industry, even to thehuman race. However, there is still no effective treatment for chicken with MD, onlyvaccine can prevent and control the occurrence of this disease.Adenovirus vector (Adv) is commonly used as an effective transgenic carrier ingene therapy and one of the most widely application prospect of transgenic carriersfor its high efficiency, high degree, low pathogenic and the advantage of not integrateinto the host cell chromosome. Our laboratory has been successfully construct therecombinant adenovirus expression vectorAd-TRAIL-2a-HN which fusion expressesTumor necrosis factor related apoptosis inducing ligand (TRAIL) gene andhemagglutinin neuraminidase (HN) gene of Newcastle disease virus by the fixed pointmutation, homologous recombination technology. This test will begin from therecombinant adenovirus positive plasmid; continue to study recombinant adenovirusAd-TRAIL-2a-HN expression in vitro and its inhibitory effect on tumor cell growth,to lay the foundation of exploring a better gene therapy for the animal tumor diseases.This study use the E.coli DH5αstrains saved at-80℃which containedrecombinant adenovirus plasmid, through kanamycin resistance screening to recoverythe positive strains, and extract the recombinant plasmid, PCR identification andsequencing analysis. To transfect theAD293cells with recombinant adenovirusplasmid for packaging which linearized by restriction enzyme PacⅠthroughlipofectin transfection, harvest infectionAD293cells can cause cell pathologicalchanges, and through fluorescence microscopy and cytotoxicity test to detect themorphology changes after AD293cells were transfected with recombinant adenovirus,RT-PCR method to detect the replication of recombinant adenovirus in transfectioncells, the results show that this study can get infectious recombinant adenovirus, wecan observe theAd-GFP expression inAD293cells under fluorescence microscope.Purify recombinant adenovirus by cesium chloride density gradient centrifugation, and detect the titer by the method of50%tissue culture infection dose (TCID50), thetiler ofAd-TRAIL, the Ad-HN,Ad-TRAIL–2A–HN andAd-GFP are108.68TCID50/mL、108.94TCID50/mL、108.28TCID50/mL�'�108.86TCID50/mL, attained thetitler for test.After transfected B16cells in vitro for48h, recombinant adenovirusreplication in cells can be detected by RT-PCR, and Western bloating can detecte theexpression of foreign genes. The result shows that the recombinant adenovirus caninfection cultured cells in vitro, and can replicate and express in the cells. ChickenMD tumor cells MDCC-MSB-1were transfected with the recombinant adenovirus invitro, the cell growth inhibition rate after transfection1-7days was detected by MTTmethod, detect the apoptosis of MSB-1by flow cytometry. The results show that therecombinant adenovirus Ad-TRAIL-2a-HN can inhibit the growth of cells and reachthe peak of31%at the third day, flow cytometry results show that the recombinantadenovirus can promote cell apoptosis rate to11.61%. The results of this study showthat infectious recombinant adenovirus can be get through the packaging and purifiedof recombinant adenovirus plasmid, and can express in cultured cells in vitro, at thesame time, the adenovirus can inhibit the growth of the MSB–1and promote the theMSB-1cell apoptosis.This study confirmed that the recombinant adenovirus can inhibit the growth ofthe MSB-1cells and induce the apoptosis of tumor cells partly in vitro through thetest of inhibition of recombinant adenovirusAd-TRAIL-2A-HN to MD tumor cellsMDDC-MSB-1. This research provided both theorial and experimental basis forinducing tumor cell apoptosis both in vitro and in vivo by using recombinatedadenovirus, and developing a wide spectrum vaccine for target therapy method foranimal tumor diseases.