牦牛IFN-α基因的克隆表达及抗病毒活性研究  

Cloning and expression of IFN-α gene of yak and its effect on antiviral activities

作　　者：蔡进忠[1] 史喜菊[2] 李少勇[2] 韩雪[2] 汪明[2] 

机构地区：[1]青海省畜牧兽医科学院,青海西宁810003 [2]中国农业大学动物医学院,北京海淀100094

出　　处：《中国兽医杂志》2006年第10期3-6,共4页Chinese Journal of Veterinary Medicine

摘　　要：从我国地方品种环湖型牦牛和野牦牛基因组DNA中克隆了α干扰素（BoIFN-α）基因．PCR产物酶切后与载体pQE30连接，构建重组质粒pQE30／BoIFN-α，将其转化JM109 E．coli并用IPTG进行诱导表达。序列分析表明．环湖型牦牛和野牦牛IFN-α成熟肽基因全长均为498个核苷酸，编码166个氨基酸，其中环湖型牦牛IFN-α与已报道的8个BoIFN-α亚型氨基酸的一致性为92．8%～95．2％．野牦牛为88．6％～92．8％，均为BoIFN-α新亚型。SDS-PAGE和Western blot结果表明．重组蛋白约为20kD，以包涵体形式存在，表达量占菌体总蛋白的25％。经包涵体提取、尿素变性和复性后，重组牛IFN-α粗提物在MDBK／VSV和CEF／VSV细胞系上的抗病毒活性分别为1．6～1．8×10^6U／mg和2．3×10^5U／mg，而且对牛传染性鼻气管炎病毒（IBRV）也有一定的抑制作用。Bovine IFN-α genes were cloned from genomic DNAs of Chinese native bovine lines （HuanHu yak and Wild yak） by PCR, and their PCR products were digested with Spk Ⅰ and Hind Ⅲ,and then was ligated to pQE30 vector similarly digested to construct recombinant expression plasmid pQE30/ BoIFN-α. The recombinant plasmid was transformed into JM109 E. coli and was induced by IPTG. Sequence analysis showed that both of the clones were composed of 498 bp encoding a mature peptide of 166 amino acids and were defined as two new subtypes with 92. 8～95. 2% and 88. 6～92. 8% （HuanHu yak and Wild yak, respectively） of amino acid sequence identities with previously published eight BoIFN-α subtypes. SDS-PAGE and Western blot analysis showed that the two recombinant proteins were inducibly expressed with molecular weight of 20 kD as inclusion bodies at a level about 25% of total bacterial proteins. The inclusion bodies were denatured and renaturated with ureas and antiviral activities of rBoIFN-α were 1. 6～ 1. 8×10^6 U/mg and 2. 3 × 10^5 U/mg in MDBK/VSV and CEF/VSV cell lines, respectively. The two recombinant proteins could also protect against IBRV challenge in the MDBK cell line.

关 键 词：牦牛 Α干扰素 抗病毒活性 

分 类 号：Q786[生物学—分子生物学]