重组犬α2干扰素的原核表达及其抗病毒活性评价  

Prokaryotic Expression of Recombinant Canine Interferon Alpha 2 and Evaluation of Their Antiviral Activities

作　　者：宋天琪[1] 金红岩[2] 张通明 陈美荣 朱鸿飞[1] 贾红[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]西藏职业技术学院,拉萨850030 [3]南京农业大学动物医学院,南京210014

出　　处：《中国畜牧兽医》2018年第1期14-21,共8页China Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项经费项目(201303042);国家重点研发计划项目(2016YFD0501003)

摘　　要：试验旨在构建犬α2干扰素(CaIFN-α2)大肠杆菌表达系统,并进行优化和筛选,评价其表达的重组犬α2干扰素的抗病毒活性。根据GenBank中CaIFN-α2的基因序列,按大肠杆菌密码子偏好性对CaIFN-α2全基因序列进行优化与合成,连接至pET-32a表达载体中,构建pET-32a-CaIFN-α2重组表达质粒。设计1对含NdeⅠ/BamHⅠ酶切位点的特异性引物,克隆CaIFN-α2成熟肽基因,连接至pET-21a表达载体中,构建pET-21a-CaIFN-α2重组表达质粒。将两种重组表达质粒转化至大肠杆菌BL21感受态细胞中,经IPTG诱导表达。SDS-PAGE结果显示,表达产物均以包涵体形式存在。表达菌经超声破碎、变性、复性和分子筛层析后,得到纯化的CaIFN-α2蛋白,纯度约为90%。采用细胞病变抑制法在MDCK-VSV系统中测定其抗病毒活性,结果显示,纯化后pET-32a-CaIFN-α2蛋白的抗病毒活性为1.5×103IU/mL,而纯化后pET-21a-CaIFN-α2蛋白的抗病毒活性高达1.0×107IU/mL。本试验结果表明CaIFN-α2在pET-32a及pET-21a载体系统中均能成功表达,且具有抗病毒活性,但在pET-21a载体系统中表达产物的活性更高,试验结果为进一步开发和利用犬干扰素制剂奠定了基础。The study was aimed to construct,optimize and screen E.coli expression system for canine interferon alpha 2（CaIFN-α2）,and evaluate the antiviral activities of the recombinant CaIFN-α2.In this study,according to the CaIFN-α2 gene sequence in GenBank,and based on the codon preferences of E.coli,the full genetic sequence of CaIFN-α2 was optimized and synthesized,then,CaIFN-α2 was connected to pET-32 ato build the pET-32 a-CaIFN-α2 recombinant expression plasmid.Therefore,one pair of specific primers with NdeⅠ/BamH Ⅰwas designed,and CaIFN-α2 mature peptide gene was cloned and connected to pET-21 ato build the pET-21 aCaIFN-α2 recombinant expression plasmid.These two recombinant expression plasmids were transformed into the E.coli BL21,then were inducted by IPTG.The results of SDS-PAGE showed that these two expression products were in inclusion body form.The recombinant CaIFN-α2 proteins were purified by ultrasonically broken,denaturation,reversion and molecular sieve chromatography respectively,and the purity was about 90%.Their antiviral activities weremeasured by inhibition method in MDCK/VSV system.The results showed that the biological activity of the recombinant CaIFN-α2 expressed in pET-32 asystem was 1.5×103 IU/mL,while the biological activity of the recombinant CaIFN-α2 expressed in pET-21 asystem was as high as1.0×107 IU/mL.These results showed that the biological activity of the recombinant CaIFN-α2 expressed in pET-21 asystem was significantly higher than the recombinant CaIFN-α2 expressed in pET-32 asystem,which laid a foundation for the further development and utilization of the recombinant CaIFN-α2.

关 键 词：犬α2干扰素(CaIFN-α2) 原核表达 纯化 抗病毒活性 

分 类 号：S852.4[农业科学—基础兽医学]