Construction Of A Recombinant Newcastle Disease Virus Expressing The Apoptin Of Chicken Anemia Virus And Its Anti-tumor Effects

As an oncolytic virus, the anti-tumor effects of Newcastle disease virus (NDV) was first reported as early as the 1950's. Cytokines induced by NDV infection, such as interferon, interleukin, tumor necrosis factor and so on; promote lymphocyte differentiation and alter the immunogenicity of tumor cells.Apoptin, the VP3 protein of chicken anemia virus (CAV), can induce apoptosis in chicken thymocyte cell, lymphoid cell line and human myeloma cell. Furthermore, apoptosis induced by apoptin is p53-independent and cannot be blocked by Bcl-2 (a proto-oncogene, inhibited apoptosis). Thus, apoptin is a potential agent for the treatment of cancers, including those lacking p53 and/or overexpressing Bcl-2.Applying the reverse genetic techniques, we inserted the apoptin gene of CAV into the genome of NDV strain ZJ-1 to generate a recombinant virus for cancer therapy. The anti-tumor effects of recombinant virus were conducted in vitro and in vivo.1 Construction of a recombinant NDV expressing the apoptinThe apoptin gene of CAV was amplified by PCR, and the fragment spanning F and M genes of the NDV ZJ1 strain was cloned. By a serial steps, the apoptin gene was inserted into the full-length cDNA clone of NDV to generate the plasmid pNDV-VP3.The pNDV-VP3, and the helper plasmids (pCI-NP, pCI-P and pCI-L), were co-transfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated specific-pathogen-free (SPF) chicken eggs, recombinant NDV was successfully rescued. Western-blot was used to confirm the expression of apoptin.2 Anti-tumor effects of a recombinant NDV expressing the apoptinFor comparison in vitro anti-tumor effects, the mortality of tumor cells A549 and SMMC7721 was measured with MTT assay. Datas were demonstrated that, following the increasing viral infective dose, mortality of tumor cells caused by NDV-ZJ1 and recombinant NDV was increasing. Results clearly indicated that at the same MOI, mortality of tumor cells caused by recombinant NDV was higher than NDV-ZJ1. Female BALB/ c mice (4-5 weeks) were used for studies of in vivo anti-tumor effects. Each of mice was subcutaneously injected with SMMC7721 tumor cells (1×108) in the abdomen. When the tumor lump reached about 200 mm3 in volume, recombinant NDV or NDV-ZJ1 was injected into the tumor lump. The tumor volume was measured every five days with a vernier caliper, and calculated on the equation. A slope of tumor volume-time curve was shown to represent in vivo anti-tumor effects of virus. Results demonstrated the tumor growth was significantly inhibited by NDV-ZJ1 and recombinant NDV in SMMC7721 tumor-bearing mice.