ISG15蛋白泛素结合酶UBCH6的原核表达及多克隆抗体制备  

Prokaryotic Expression of ISG15 Protein Ubiquitin-binding Enzyme UBCH6 and Preparation of Polyclonal Antibodies

作　　者：李玉霄 唐榕泽 高跃美 冯佳佳 李晓泉[1,2] 梁晶晶 李晓宁[1,2] 罗廷荣 LI Yu-xiao;FENG Jia-jia;TANG Rong-ze;GAO Yue-mei;LI Xiao-quan;LIANG Jing-jing;LI Xiao-ning;LUO Ting-rong(Laboratory of Animal Infectious Diseases,College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院动物传染病研究室,广西南宁530004 [2]亚热带农业生物资源保护利用国家重点实验室,广西南宁530004

出　　处：《动物医学进展》2020年第12期28-33,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31660722);广西自然科学基金项目(2017GXNSFAA198137);国家重点研发计划项目(2018YFD0500104)。

摘　　要：通过抽提接毒猪瘟病毒(CSFV)的PK-15细胞RNA,以RT-PCR方法克隆UBCH6基因,并与pMD18-T载体连接,再通过双酶切法将UBCH6基因与原核表达载体pGEX-4T-1、真核表达载体pcDNA3.0连接。重组原核表达载体pGEX-UBCH6用感受态细胞BL21转化,并在低温条件下,用低浓度的IPTG诱导表达UBCH6重组蛋白,经SDS-PAGE电泳和考马斯亮蓝染色结果显示,在30℃、用0.05 mmol/L IPTG诱导时UBCH6表达效果最好,而且UBCH6重组蛋白主要以可溶性的形式在菌液中表达。UBCH6重组蛋白经过Ni-NTA纯化后与弗氏完全佐剂、弗氏不完全佐剂等体积混合乳化并免疫4周龄昆明小鼠,从而制备出UBCH6多克隆抗体,重组真核表达载体pcDNA3.0-UBCH6以化学转染人工脂质体法导入293T细胞中,Western blot检测制备的UBCH6多克隆抗体能与真核表达细胞蛋白发生反应,反应效价可达1∶10000,反应性良好。Western blot检测UBCH6多克隆抗体的特异性,ISG15多克隆抗体、UBCH6多克隆抗体都能与接毒CSFV、转染PolyI:C的PK-15细胞蛋白发生良好反应,且蛋白表达量与时间呈正相关。The UBCH6 gene was cloned by RT-PCR and extracted into pMD18-T vector by double-enzyme digestion.The UBCH6 gene and prokaryotic expression vector pGEX-4 T-1 were isolated by double enzyme digestion.The eukaryotic expression vector pcDNA3.0 was ligated.The recombinant prokaryotic expression vector pGEX-UBCH6 was transformed into competent cell BL21,and the recombinant protein of UBCH6 was induced by low concentration of IPTG under low temperature conditions.The results of SDS-PAGE and Coomassie blue staining showed that the expression of UBCH6 was best at 30℃,IPTG 0.05 mmol/L,and the UBCH6 recombinant protein was mainly expressed in the bacterial solution in a soluble form.UBCH6 recombinant protein was purified by Ni-NTA and emulsified in the same volume with Freund's complete adjuvant and Freund’s incomplete adjuvant and immunized in 4 weeks old Kunming mice to prepare UBCH6 polyclonal antibody,and recombinant eukaryotic expression vector pcDNA3.0-UBCH6 was introduced into 293 T cells by chemical transfection of artificial liposome,and detected by Western blot method.The prepared UBCH6 polyclonal antibody can react with eukaryotic expression protein,and the reaction titer can reach 1∶10 000.The specificity of UBCH6 polyclonal antibody was detected by Western blot.Both ISG15 polyclonal antibody and UBCH6 polyclonal antibody reacted well with CSFV and PK-15 cell protein transfected with PolyI∶C,and the protein expression and time showed positive correlation.

关 键 词：ISG15蛋白 UBCH6基因 多克隆抗体 猪瘟病毒 蛋白诱导表达 

分 类 号：S852.43[农业科学—基础兽医学] S852.651[农业科学—兽医学]