比格犬α7干扰素成熟肽基因的原核表达及表达产物活性的分析  

Prokaryotic expression of Beagle IFN-α7gene and analysis of its antiviral activities

作　　者：潘福星[1] 朱梅胜 冯培祥 王冬冬[1] 范丹丹[1] 齐娟 王祖荣 尹燕博[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]青岛澳兰百特生物工程有限公司,山东青岛266101 [3]青岛博隆实验动物有限公司,山东青岛266225

出　　处：《中国兽医科学》2014年第3期298-302,共5页Chinese Veterinary Science

基　　金："十二五"农村领域国家科技计划项目(2012AA101303)

摘　　要：从比格犬的脾中提取基因组DNA,采用RT-PCR方法扩增比格犬α7干扰素成熟肽基因。将纯化的PCR产物克隆至pMD19-T载体,转化入感受态细胞DH5α中,PCR检测及测序鉴定结果表明,克隆的目的基因为比格犬α7干扰素成熟肽,与GenBank中登录的犬干扰素序列(NM001006654)的同源性达到100%。在此基础上构建重组原核表达质粒pET-32a-CaIFN-α7,并转化至表达宿主BL21(DE3)中。该工程菌在37℃经IPTG诱导后进行SDS-PAGE电泳,结果在分子质量约38.4ku处出现了目的蛋白条带,表达产物主要以包涵体形式存在。经Bandscan软件分析,表达产物约占菌体总蛋白的48.5%。用Protein Refolding Kit对包涵体进行纯化复性,重组CaIFN-α7在犬肾细胞(MDCK)上具有较高的抗病毒活性。The genomic DNA was extracted from dog^s spleen and used as the template for amplifica- tion of the mature peptide gene of CaIFN-α7 gene by PCR. The purified PCR product was cloned into pMD19-T vector,and transformed into DHSa competent cells,the detection by PCR and sequencing showed that the cloned gene contained the full nucleotide sequence of Beagle＇s IFN-α7,and the sequence alignment showed that the PCR-amplified gene was identical to the published sequence in GenBank（NM001006654）. The recombinant prokaryotic expression vector pET-32a-CaIFN-α7 was constructed, and transformed into the expression host BL21（DE3）. After induction with ITPG at 37 ℃ ,a fusion protein with an expected size of about 38.4 ku was detected and existed in the form of inclusion bodies by SDS-PAGE. The Bandscan a- nalysis indicated that the fusion protein accounted for 48.5 % of the total bacterial protein. The target pro- tein body was refolded and purified by Protein Refolding Kit,and CaIFN-α7 had high antiviral activities in canine cell MDCK. This study laid foundation for further explore the production and application of CalFN-α7.

关 键 词：比格犬 α7干扰素成熟肽基因 克隆 原核表达 抗病毒活性 

分 类 号：S852.42[农业科学—基础兽医学]