重组鲁西黄牛α干扰素融合蛋白的表达及其抗病毒活性研究

Expression，Purification of Recombinant Luxi Yellow Cattle IFN-α Fusion Protein and Its Antiviral Activities

摘要:

通过PCR从鲁西黄牛(Yellow cattle)基因组DNA中克隆了α干扰素(BoIFN-α)基因，并插入到pET32a+中, 构建成重组原核表达质粒pET32a+/BoIFN-α，进行测序和诱导表达。测序结果表明，鲁西黄牛IFN-α基因全长498个核苷酸,含一个开放阅读框(ORF),编码166 个氨基酸的成熟蛋白，与已报道的牛α干扰素C亚型氨基酸组成同源性为97.6%。表达产物经SDS-PAGE分析，表达出40kD的融合蛋白，表达量占菌体总蛋白的26.7%。表达产物经镍离子螯合次氨基三乙酸(Ni-NTA)亲和层析纯化，纯化产物进行复性后在MDBK/VSV上的活性为5×10⁵u/mg。重组牛IFN-α（rBoIFN-α）对牛轮状病毒(BRV) 有一定的抑制作用,抗BRV病毒活性为1.5×10⁵u/mg。结果显示从鲁西黄牛中克隆了IFN-α基因的一种新亚型,即BoIFN-α C2，并实现了高效表达，获得了具有较高抗病毒活性的重组干扰素产物，为重组牛干扰素的开发奠定了基础。

Abstract:

Interferon α 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-α. The expression of BoIFN-α in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-α gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-α 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 40kD 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 5×10⁵u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-α C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.