重组猪α干扰素/白细胞介素-2的可溶性表达及体外活性研究  

The Soluble Expression and Bioactivity of Porcine(Sus scrofa)Recombinant Interferonα/Porcine Interleukin-2 In vitro

作　　者：冯桂丹 闫若潜[2,3] 马震原 柴茂[2,3] 周建浩 康台生 王淑娟 杨海波 刘影[2,3] 谢彩华 赵雪丽[2,3] 王翠 FENG Gui-Dan;YAN Ruo-Qian;MA Zhen-Yuan;CHAI Mao;ZHOU Jian-Hao;KANG Tai-Sheng;WANG Shu-Juan;YANG Hai-Bo;LIU Ying;XIE Cai-Hua;ZHAO Xue-Li;WANG Cui(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Henan Centre for Animal Diseases Control and Prevention,Zhengzhou 450008,China;Key Laboratory of Surveillance and Early Warning,Prevention and Control of Major Animal Diseases of Henan Province,Zhengzhou 450008,China;Shanghai Animal Disease Prevention and Control Center,Shanghai 201103,China;College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China)

机构地区：[1]河南农业大学动物医学院,郑州450002 [2]河南省动物疫病预防控制中心,郑州450008 [3]河南省重大动物疫病监测预警与防控重点实验室,郑州450008 [4]上海市动物疫病预防控制中心,上海201103 [5]河南科技大学动物科技学院,洛阳471000

出　　处：《农业生物技术学报》2024年第3期595-604,共10页Journal of Agricultural Biotechnology

基　　金：河南省重点研发专项(231111111300):河南省重大科技专项(221100110600);河南省现代农业产业技术体系(HARS-22-12-T);河南省科技攻关项目(232102110089)。

摘　　要：猪(Sus scrofa)α干扰素/白细胞介素-2(porcine interferonα/porcine interleukin-2,PoIFN-α-linkerPoIL-2)在大肠杆菌(Escherichia coli)中多以包涵体形式表达,为在大肠杆菌系统中获得可溶性表达的具有活性的重组猪α干扰素/白介素2(rPoIFN-α-linker-PoIL-2)蛋白,本研究根据大肠杆菌密码子偏好性对PoIFN-α-linker-PoIL-2嵌合基因进行可溶性改造并合成。将改造后的PoIFN-α-linker-PoIL-2嵌合基因克隆到表达载体pET-32a(+)进行原核表达,采用镍铬亲和层析柱对表达的可溶性rPoIFN-α-linker-PoIL-2蛋白进行纯化。分别采用淋巴细胞增殖检测法、ELISA方法检测rPoIFN-α-linker-PoIL-2蛋白体外促猪外周血T淋巴细胞(peripheral blood T lymphocyte,PBLC)增殖活性,及其与抗猪白介素2单抗、抗猪α干扰素单抗发生特异性免疫反应的活性;采用细胞病变抑制法检测rPoIFN-α-linker-PoIL-2蛋白在不同细胞系上抑制不同病毒的增殖活性。结果显示,PoIFN-α-linker-PoIL-2嵌合基因可在大肠杆菌中高效可溶性表达,表达的rPoIFN-α-linker-PoIL-2蛋白分子量大小约55 kD。rPoIFN-α-linker-PoIL-2蛋白经镍铬亲和层析柱纯化后纯度达90%以上。rPoIFN-α-linker-PoIL-2具有显著的促PBLC增殖活性(P<0.05);可以与抗PoIL-2、PoIFN-α单抗发生特异性免疫反应。rPoIFN-α-linker-PoIL-2蛋白在不同细胞上抑制不同病毒的增殖活性效价有差异,在猪肾细胞(porcine kidney cells,PK)-15、人羊膜细胞(human amniotic cells)WISH上抑制水泡性口炎病毒(Vesicular stomatitis virus,VSV)增殖的活性单位分别为1.8×10^(6)和2.5×10^(6) IU/mg;在PK-15细胞上抑制伪狂犬病毒(Pseudorabies virus,PRV)、塞内卡病毒(Seneca virus A,SVA)增殖的活性单位分别为2.2×10^(4)、1.3×10^(5) IU/mg;在非洲绿猴肾细胞(Verda Reno,Vero)上抑制猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)增殖的活性单位为3.2×10^(4) IU/mg。本研究获得了可溶性表达的rPoIFN-α-linkerPoIL-2蛋�Porcine(Sus scrofa)interferonα/interleukin-2(PoIFN-α-linker-PoIL-2)is mostly expressed as inclusion bodies in Escherichia coli.To obtain soluble expression of active recombinant(r)PoIFN-α-linkerPoIL-2 in the E.coli system,the chimeric gene PoIFN-α-linker-PoIL-2 was synthesised by soluble modification based on the codon preference of E.coli.The modified PoIFN-α-linker-PoIL-2 chimeric gene was cloned into the expression vector pET-32a(+)for prokaryotic expression,and the expressed soluble recombinant fusion protein(rPoIFN-α-linker-PoIL-2)was purified using a nickel-chromium affinity chromatography column.The proliferative activity of rPoIFN-α-linker-PoIL-2 protein on peripheral blood T lymphocytes in vitro was detected by lymphocyte proliferation assay,could also be detected by ELISA assay using anti-PoIL-2 monoclonal antibody or anti-PoIFN-αmonoclonal antibody.The antiviral bioactivity of rPoIFN-α-linker-PoIL-2 protein was tested by inhibiting the 50%appearance of cytopathic effect(CPE)of different viruses on different cell lines.The results showed that the chimeric gene PoIFN-α-linker-PoIL-2 could be efficiently expressed in E.coli.The expressed rPoIFN-α-linker-PoIL-2 protein had a molecular weight of about 55 kD.The purity of rPoIFN-α-linker-PoIL-2 protein was over 90%after purification by NiCr affinity chromatography,which had significant proliferative activity on peripheral blood T lymphocytes(PBLC).Specific immune response can be detected by anti-PoIL-2 and anti-PoIFN-αmonoclonal antibodies.The rPoIFN-α-linker-PoIL-2 protein had different inhibitory activities on the proliferation of different viruses in different cells.The activity units of inhibiting the proliferation of Vesicular stomatitis virus(VSV)in porcine kidney cells(PK-15)and human amniotic cells WISH were were 1.8×10^(6) and 2.5×10^(6) IU/mg.The active units of inhibiting the proliferation of Pseudorabies virus(PRV)and Seneca virus(SVA)on PK-15 cells were 2.2×10^(4) and 1.3×10^(5) IU/mg,respectively.The activity unit for inhibiti

关 键 词：猪Α干扰素 猪白细胞介素-2 嵌合基因 可溶性表达 体外活性 

分 类 号：S852.4[农业科学—基础兽医学] S859.79[农业科学—兽医学]