Construction of a Retroviral Vector for Production of Transgenic Cells Expressing Feline Immunodeficiency Virus (FIV) Viral Protein

Abstract

Retroviral vectors are being used as vehicles for transporting foreign genes into mammalian cells for the purpose of human gene therapy to treat viral disease like human immunodeficiency virus (HIV). In efforts to develop a preventative vaccine against a lentiviral infection, research is centering on developing a protective vaccine that will identify antigens which induce protective cell mediated immunity, especially cytotoxic T lymphocytes (CTL). Retroviral vectors provide the required endogenous expression of a retrovirus antigen for identificatoin of CTL responses against that individual antigen. Feline immunodeficiency virus (FIV) is an animal lentivirus that shares common clinical properties with HIV. The commonalities shared between HIV and FIV allows the use of FIV as an animal model for developing a protective vaccine that will identify antigens which induce protective cell mediated immunity or CTL responses. The FIV gag nucleocapsid (p10) protein will be cloned into the retroviral vector pLXSN for production of transgenic cells expressing the p10 protein for study of specific T-cell responses to this FIV antigen. PCR will be used to amplify the nucleocapsid from the pUC119 plasmid containing the FIV genome. The amplified p10 PCR product will be EcoRI and BamHI digested for directional insertion into the EcoRI and BamHI digested retroviral vector pLXSN. Transformation of competent E. coli cells will provide colonies containing the recombinant retroviral vector DNA. Analysis of the recombinant retroviral vector DNA will be carried out by PCR. Amplification by PCR of the nucleocapsid protein provided the p10 DNA sequence. Subsequent EcoRI and BamHI digestion revealed a natural EcoRI site at base pair 1871, therefore cutting off 160 bp from the p10 sequence. The upstream primer was reconstructed with an XhoI cut site for reamplification of the p10 protein from the pUC119 plasmid and will be inserted into the retroviral vector by XhoI and BamHI digestion. The 204 bp sequence generated from the EcoRI and BamHI digestion was inserted into the retroviral vector and awaits analysis of the idetification of recombinant retroviral vector colonies.