牛源ISG15蛋白的原核表达及抗血清的制备  

PROKARYOTIC EXPRESSION OF ISG15 PROTEIN AND PREPARATION OF ANTI-ISG15 POLYCLONAL ANTIBODIES

作　　者：陶洁[1,2,3] 廖金虎[1,2] 张倩[1,2] 张信军[1,2] 朱国强[1,2] 

机构地区：[1]扬州大学兽医学院,扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [3]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《中国动物传染病学报》2015年第2期68-73,共6页Chinese Journal of Animal Infectious Diseases

基　　金：江苏高校优势学科建设工程资助项目;重大动物疫病防控技术引进(国家农业部948计划;2011-G24);江苏省属高校自然科学重大基础研究项目(14KJA230001);江苏省农业支撑项目(BE2014358);泰州市农业科技计划项目(TZ201421)

摘　　要：本研究利用人重组干扰素IFNα-2A刺激牛肾细胞(Mardin-Darby bovine kidney cells,MDBK),并提取细胞总RNA。RT-PCR扩增牛源干扰素刺激基因15(interferon-stimulated gene 15,ISG 15),将其克隆入p Cold-TF表达载体中,转化大肠杆菌BL21(DE3)。经IPTG诱导表达后,获得约70 k Da可溶性ISG15重组蛋白。用试剂盒对重组蛋白ISG15进行纯化回收,经BCA法测定其蛋白浓度为1 mg/m L。用纯化的ISG15重组蛋白免疫ICR小鼠,制备多克隆抗体血清,用间接ELISA方法测定多抗血清效价为1:102 400。利用Western blot进一步鉴定ISG15多抗血清,结果发现其与纯化的ISG15重组蛋白能发生特异性反应,可用于后续研究。In the present study, total RNA was extracted from MDBK cells stimulated with recombinant human interferon IFN α-2A and interferon-stimulated gene 15 （ISG15） was amplified using RT-PCR. Then, ISG15 gene was cloned into prokaryotic expression vector pCold-TF and transformed into E.coli BL21 （DE3） competent cells. Soluble ISG 15 protein with molecular mass of 70 kDa was expressed following induction with IPTG and purified. The concentration of the purified ISG15 was lmg/mL as measured in BCA kit. Subsequently, ICR mice were inoculated with the purified ISG15 for preparation of polyclonal antibodies. Blood samples were collected from mice post the fourth immunization and antibody titer was 1：102 400 as detected in indirect ELISA. The reactivity of antiserum preparation to ourified ISG 15 was confirmed in Western blot.

关 键 词：ISG15 蛋白表达 多抗血清 

分 类 号：S852.4[农业科学—基础兽医学]