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

作　　者：张永红[1] 王长法[1] 杨少华[1] 高运东[1] 王洪梅[1] 李景鹏[2] 仲跻峰[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,济南250100 [2]东北农业大学生命科学学院,哈尔滨150030

出　　处：《生物工程学报》2007年第4期730-734,共5页Chinese Journal of Biotechnology

基　　金：山东省农科院青年基金(No2005YQ036);山东省农科院高技术自主创新基金(No2006YCX027);山东农科院重大成果培育计划项目(No2006YCG012)资助~~

摘　　要：通过PCR从鲁西黄牛(Yellowcattle)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-αC2,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。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 × 10^5 u/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 coll. Moreover the purification had good effects on antiviral activities.

关 键 词：黄牛 α干扰素基因 表达 抗病毒活性 

分 类 号：R91[医药卫生—药学]