Establishment Of Cell Line For E1-deleted PAdV-3 Virion Rescue

Porcine adenovirus 3(PAdV-3) was firstly isolated from healthy pig intestinal tract in 1964. For the study of porcine oral vectored vaccine based on adenovirus, PAdV-3 is superior in aspects of immunogenicity, intestinal tropism and evasion self-vector immunity to human adenovirus type 5(HAdV-5) which has been widely used as vehicle for gene therapy and vaccine study. For human vaccine study, as there is no cross-immunity between PAdV-3 and HAdV-5, the vectored vaccines based on PAdV-3 can avoid the interference and tissue toxicity caused by HAdV-5 immunity.To make full use the aforementioned advantages of PAdV-3, E1-deleted PAdV-3(PAdV-3 ΔE1) infectious clone and high-efficient package cell line are two prerequisite. The PAdV-3 ΔE1 was kindly donated by Dr. Suresh Tikoo in VIDO-InterVac, Canada. However, the only reported PAdV-3 ΔE1 package cell line, namely VR1 BL is protected by 13 patents, which limits the vaccine study based on PAdV-3. As wild-type PAdV-3 can proliferate efficiently in swine testicular(ST) cells and VR1 BL cells, we established a novel cell line for PAdV-3 ΔE1 rescue and replication based on ST cells. The novel cell line(ST-E1-E1Blarge-ISceI) can express PAdV-3 E1 Blarge and I-SceI stably. Compared with VR1 BL cells, ST-E1-E1Blarge-ISceI cells could also rescue PAd V-3 ΔE1 efficiently although the efficiency was little lower than VR1 BL cells. However, the viral proliferation efficiency was about 100 times higher than VR1 BL cells. The contents and results are as follows:1. Production of the secondary recombinant lentivirusTo construct clone plasmid of the secondary generation lentivirus, 3NLS-ISceI and His-E1 Blarge gene were inserted into MCS1 and MCS2 of cloning plasmid pTrip-CMV-3HAMCS1-2A-MCS2-IRES-Hygro respectively. Plasmid pTrip-CMV-3HA-3NLS-ISceI-2A-HisE1Blarge-IRES-Hygro and two common plasmids, pMD2.G(expression viral envelop protein) and psPAX2(expression viral package proteins), were co-transfected into 293 T cells for lentivirus production. The lentivirus, Lenti-3HA-ISceI-2A-His-E1Blarge-IRES-Hygro, was collected and titrated on 293 T cells by TCID50/mL. Then antibiotic selection and western blot were used to validate the accuracy of lentivirus.2. Selection of ST-E1-E1Blarge-ISceI cellsST-E1 cells were infected by the lentivirus in 10 MOI. Then the cells were selected by hygromycin. The positive cell clones were picked out and spreaded over 30 passages, the samples of passage 5, 10 and 30(P5, P10, P30) were collected. The transgenes expression was tested by western blot using anti-HA mouse monoclonal antibody and anti-His mouse monoclonal antibody. In summary, ST-E1-E1Blarge-ISce I cells were selected successfully and can express PAdV-3 E1 Blarge and intron-encoded endonuclease(I-SceI) stably.3. Comparison the difference between VR1 BL and ST-E1-E1Blarge-ISceI cells in PAdV-3E1 rescue and propagationPAdV-3 E1 genome was linearied by Pac I, and transfected VR1 BL cells and ST-E1-E1Blarge-ISceI cells using Lipofectin reagent to rescue PAdV-3 E1 virions. The virus rescue efficiency in VR1 BL and ST-E1-E1Blarge-ISceI cells was compared by transfection efficiency and TCID50/mL. The results showed that ST-E1-E1Blarge-ISceI cells can rescue PAdV-3 ΔE1 successfully also the efficiency of viral genome transfection and virions rescue was a little lower than VR1 BL cells. In the aspect of viral proliferation, our eatablished cells had similar efficacy with VR1 BL cells within 72 h. However, the viral proliferation titer in our established cells was 100 times higher than that in VR1 BL cells within 96 h, as near 50% of our established cells still survived for viral proliferation, while viral infected VR1 BL cells were completely detached and died from 72 h to 96 h.In summary, this novel cell line can package and proliferate PAdV-3 ΔE1 efficiently. Our established cell line could greatly facilitate the research of oral vaccine and gene delivery based on PAdV-3 ΔE1.