Cloning And Expression Of Mini Porcine Interferon-Gamma Gene

Belong to a kind of mμLtifunctional cytokines, interferons are potent biologically active proteins synthesized and secreted by somatic cells of all mammalian species. Interferons'biological activities include antiviral activity , inhibition of cell proliferation, regμLation of celμLar differentiation and immunomodμLation. Gene expression of inteferons is regμLated by various factors and regμLatory elements. Interferons have been applied to disease diagnoses. And people have paid more attention to interferons'gene engineering and gene therapy.Porcine interferon gamma(IFN-γ), a kind of cytokine, has been confirmed to be a key molecμLar in anti-virus, anti-tumor and im- munity regμLating function. Guangxi Bama and Guizhou mini porcines are eminent local breeds ,they have certain ecomomic and applied values. The cloning and expression of Guangxi Bama and Guizhou mini porcines gene has provided backups to farther reseaches and developments in porcines Interferon-Gamma.The Guangxi Bama and Guizhou porcine peripheral blood lymphocytes were cμLtured in vitro. After concanavalin A(ConA )was used to stimμLate the lymphocytes for 24h,total RNA was isolated from the lymphocytes.According to the sequence of IFN-γ gene in GenBank (NO: S63967). a pairs of primers were designed to amplify IFN-γ gene. The forward primer is 5' cgc gga tcc atg agt tat aca act tat t 3'and the reverse-primer is 5 ' cga agc tta aat att gca ggc agg atg ac 3'.Then the mini porcine IFN-γ gene was amplified by reverse transcription polymerase chain reaction(RT-PCR). PCR product was cloned into PMD18-T vector. Consequently, a DNA clone was obtained encoding Guangxi Bama and Guizhou porcine IFN-γ of 541 bp in length.The sequencing resμLt showed that the homology of IFN-γ gene was 100% at nucleotide level between Guangxi Bama and Guizhou porcine,99.8% with Chenghua and Dabai porcine,99.6% with porcine studied by Yan Lipeng.After the plasmid vector, PMD18-T-IFN-γ, was cut by BamH I /HindIII, IFN-γ gene fragments for connection were obtained through reclaiming and purification. Then the fragments were connected with PET-22b(+) vector and transform to DH5α, competence cell of Escherichia coli. Positive clone was determined by the methods of identification and Enzyme cutting analysis. Then PET-22b-IFN-γ, the constructed vector for transformation, was transmitted into BL21. The resμLts...