The Cloning And Expression Of The Porcine Interferon-gamma Gene

Porcine interferon-gamma genomic gene was cloned by long-PCR amplification which template is genomic DNA extracting from the liver tissues of pigs. A 99% sequence homology was indentified by the construct between the Genbank porcine interferon-gamma gene and my sequenced products. Then the purified porcine interferon-gamma genomic gene was cloned into PCI-neo vector to construct eukaryotic expression vector PCI-neo-PoIFN y . The Construct was transfected into the Chinese Hamster Ovary (CHO) Cells by The lipofectamin-mediated DNA transfection procedure. After cultured with G418, transgenic cell clones which can express porcine interferon-gamma protein were rebulided.The total RNA was gained by UNIQ-10 Trizol Total RNA Extract Kit from collectted transgenic cells. Then the PoIFN Y cDNA gene was amplified by RT-PCR, and this result was also testified that the porcine interferon-gamma gene has been successfully inserted into the genomic DNA of CHO and translated its mRNA. the purified porcine-gamma cDNA gene was cloned into PBV220 vector to construct prokaryotic expression vector PBV220-cPoIFNY , and a 100% sequence homology was indentified by the construct between the Genbank porcine interferon-gamma cDNA and my two-directional sequenced products. The expression plasmid was transferred into E. coli. BL21 or DH5 a and induced by controlling tempreture. According to the detection of SDS-PAGE and Western blot analysis, The plasmid has been expressed, and the 17KD production was approximately 15% of the total protein. It was also testified that the expression of PoIFN Y protein between E. coli DH5a and BL21 has no apparently different.