水牛IFN-γ在毕赤酵母中的表达及其单克隆抗体的制备  

Expression of Bubalus bubalus IFN-γ in Pichia pastoris and Preparation of Its Monoclonal Antibodies

作　　者：邹新峰[1,2] 郭爱珍[1,2] 陈颖钰[1,3] 胡长敏[1] 王冰[1,3] 刘晓乐[1,2] 谢倩[1,2] 姜勇[1,2] 张策[1,2] 陈焕春[1,2] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [2]华中农业大学动物医学院,湖北武汉430070 [3]华中农业大学动物科技学院,湖北武汉430070

出　　处：《动物医学进展》2013年第6期71-77,共7页Progress In Veterinary Medicine

基　　金：公益性行业科研专项"布鲁氏菌病;牛结核病防控技术研究"(200903027);家畜生产运输过程中环境应激响应及调控技术研究(201003060)

摘　　要：本研究旨在建立水牛IFN-γ(Bubalus bubalus IFN-γ,Bb IFN-γ)体外释放检测法,为定量检测水牛IFN-γ、开发水牛结核病及其他疫病的检测技术奠定基础。克隆了水牛IFN-γ成熟肽基因片段,根据测序结果,在不改变氨基酸序列的情况下,将密码子替换成毕赤酵母偏爱密码子并合成全基因,进一步用毕赤酵母GS115表达了水牛IFN-γ成熟肽基因。通过水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上检测了其抗病毒活性,抗VSV活性为7.44×105 U/mL。利用酵母表达的水牛IFN-γ蛋白免疫Balb/c小鼠,并筛选阳性杂交瘤细胞,获得5株高效价的单克隆抗体。利用该重组蛋白免疫日本长耳大白兔,得到高效价的抗水牛IFN-γ多克隆抗体。用所获得的单克隆抗体及多克隆抗体建立了检测水牛IFN-γ的双抗体夹心ELISA方法,检测灵敏度达到125pg/mL。通过建立的方法分别检测了原核表达的奶牛IFN-γ、梅花鹿IFN-γ、弥猴IFN-γ以及其他7种非相关重组蛋白,结果表明所建立的方法灵敏度高,特异性好。This study was aimd to establish Bubalus bubalus IFN-γ（BbIFN-γ） detection method and lay the foundation for development of BbIFN-γ in vitro release assay to diagnose buffalo tuberculosis or other diseases.The sequence of the mature peptide of buffalo IFN-γ was cloned and sequenced.According to the Pichia pastoris codon usage bias,the sequence was changed without altering amino acid sequence and synthesized.The protein was expressed in P.pastoris GS115,and the antiviral activity to vesicular stomatitis virus（VSV） grown in Mardin-darby bovine kidney（MDBK） cells was determined to be 7.44×105U/mL.The expressed buffalo IFN-γ was used to immunize Balb/C mice and five monoclonal antibodies（McAb） to BbIFN-γ were obtained according to the conventional methods.This protein was further used to immunize the rabbits and the polyclonal antibodies（PcAb） with high titers were obtained.A double-antibody sandwich ELISA to detect BbIFN-γ was established by using the home-made McAb and PcAb,the sensitivity was determined to be 125 pg/mL.The specificity was tested by using recombinant IFN-γ of the dairy cow,deer,macaque,and 7 unrelated recombinant proteins,and the results confirmed that the specificity is good.

关 键 词：水牛 Γ干扰素 毕赤酵母 双抗体夹心酶联免疫吸附试验 单克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学]