梅花鹿IFN－α基因的克隆与原核表达研究

摘要

In order to investigate the antiviral function and molecular mechanism of interferon alpha( IFN-α) ,one pair of primers were designed and synthesized according to the bovine IFN-α gene nucleic acid sequence ( A00145) published in the GenBank. The IFN-α gene of sika deer was amplified by PCR from genome DNA extracted directionally from liver of sika deer, then cloned and sequenced. the result showed the full length sequence of sika deer IFN-αgene was consisted of 570 bp, which encode 189 amino acids, Containing 18 amino acids of signal peptide and mature polypeptide of 169 amino acids. IFN-αprotein of sika deer had one transmembrane domain. IFN-αgene was different among species due to their evolution status.The primers of IFN-αmature peptide were synthesized according to the result of sequencing. IFN-αmature peptide gene was amplified. The expression vector pET28a-IFNα was constructed and transformed into E. coli BL21 ( DE3 ) cells. By the detection of SDS-PAGE and Western blotting,the Sika deer IFN-αrecombinant protein was 24 kd and formed an insoluble inclusion body. The amount of recombinant protein accounted for 34.04% of the total fraction of bacterial proteins. Expression products could react with His tag antibody specificity reaction. The expression product of pET28a-IFNα was purified by The Ni column and renatured. Then, cytopathic inhibition method detected that express product possess antiviral activity. This study provide the foundations for study of the antiviral molecular mechanism of sika deer IFN-α and the development of antiviral drugs.%为研究梅花鹿IFN－α的抗病毒功能和分子机制，根据GenBank中牛IFN－α基因序列（A00145），设计并合成1对引物，以梅花鹿肝脏组织DNA为模板，采用PCR技术扩增梅花鹿IFN－α全基因核苷酸序列。序列分析表明梅花鹿IFN－α基因全长570 bp，编码189个氨基酸，其中含18个氨基酸的信号肽和169个氨基酸的成熟多肽，存在1个潜在跨膜区，基因随不同物种发育进化地位表现出种属间的差异性。在此基础上，合成1对引物，扩增梅花鹿IFN－α成熟肽核苷酸序列，构建pET28a－IFNα原核重组表达质粒并转化至大肠杆菌 BL21（DE3）感受态细胞中。 SDS－PAGE 和Western blotting检测结果表明，在24 kd左右出现特异性蛋白带，表达产物主要为不溶性的包涵体，表达量占菌体总蛋白的34．04％，且表达产物与抗His标签抗体可发生特异性反应。将Ni柱纯化的pET28a－IFNα诱导表达产物复性后，细胞病变抑制法检测表明表达产物具抗病毒活性。研究结果为梅花鹿IFN－α蛋白抗病毒分子机制的研究及抗病毒药物的开发奠定了基础。