鲁西黄牛α干扰素基因的原核表达及其抗血清的制备

Prokaryotic Expression of IFN-α Gene of Luxi Yellow Cattle and Preparation of Antisera Against BoIFN-α

采用PCR方法从鲁西黄牛基因组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1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40 ku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免疫昆明系小鼠2次,制备高滴度的牛IFN-α抗血清。结果从鲁西黄牛中克隆了IFN-α基因的一种新亚型,即BoIFN-αC2,实现了高效表达和纯化,并制备了鼠抗牛IFN-α,为重组牛干扰素的开发奠定了基础。

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 40 ku, and the recombinant proteins accounted for 26.7% of the whole proteins. The expressed product was puried by affinity chromatography with immobilized nickel chelating NTA（Ni-NTA）. Antisera against BoIFN-α was prepared by immunizing rat with the purified protein. The expression and purification of the recombinant BoIFN-α fusion protein and preparation of the antisera against BoIFN-α will lay a foundation for the future application of BoIFN-α.