[Expression, purification of recombinant Luxi yellow cattle IFN-alpha fusion protein and its antiviral activities].
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.
Zhang
Yong-Hong
YH
Research Center of Dairy Cattle Science, Shandong Academy of Agricultural Science, Jinan 250100, China.
Wang
Chang-Fa
CF
Yang
Shao-Hua
SH
Gao
Yun-Dong
YD
Wang
Hong-Mei
HM
Li
Jing-Peng
JP
Zhong
Ji-Feng
JF
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