title: [Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities].
abstract: Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.
authors.affiliation: Research Center of Dairy Cattle Science, Shandong Academy of Agricultural Science, Jinan 250100, China.
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Zhang
authors.forename: Chang-Fa
authors.initials: CF
authors.lastname: Wang
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Yang
authors.forename: Yun-Dong
authors.initials: YD
authors.lastname: Gao
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Wang
authors.forename: Jing-Peng
authors.initials: JP
authors.lastname: Li
authors.forename: Ji-Feng
authors.initials: JF
authors.lastname: Zhong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification*; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012401:Rotavirus / Q000187:drug effects; D017421:Sequence Analysis