稳定分泌抗牛γ-干扰素杂交瘤细胞株的建立与鉴定
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摘要
γ-干扰素(IFN-γ)主要是在特定的抗原、有丝分裂原的刺激下由活化T淋巴细胞或NK细胞产生的一类具有广谱抗病毒免疫调节等多种生物学活性的糖蛋白。国内外在牛IFN-γ的研究和应用方面的工作已经开展很多,但不论是对免疫细胞功能及机体细胞免疫应答方面的研究,还是对疾病状态下IFN-γ产生水平的判定,都需要一种简便、快速的检测手段。应用牛IFN-γ单克隆抗体作为研究、检测IFN-γ的手段,在免疫机制的研究、免疫功能检测等领域具有广泛的应用。
本研究用原核表达的重组牛IFN-γ(rHis-BoIFN-γ)免疫6周龄BALB/c雌性小鼠,免疫3次后取其脾细胞和骨髓瘤细胞SP2/0进行融合。用建立的间接ELISA方法筛选阳性杂交瘤细胞,经3~5次亚克隆,最终获得12株能稳定分泌抗体的阳性杂交瘤细胞株。亚类鉴定结果显示12株杂交瘤细胞均为IgG,其中5株为IgG1,3株为IgG2a,4株为IgG2b。12株杂交瘤细胞在体外具有稳定分泌抗BoIFN-γ单克隆抗体的能力。Western-blot及间接ELISA证明12株单抗均可特异性的结合rBoIFN-γ蛋白。通过间接免疫荧光显示,12株单抗均与真核细胞BHK21表达的rBoIFN-γ反应产生荧光反应,证明了单抗能够与真核细胞产生的BoIFN-γ反应,证实了单抗的特异性。ELISA叠加试验表明,3C2与1H4、1G6与1G4针对不同表位,1A10、1B2、2B9、3C4 4株和1C1与1G6、1G4与3C2、1A10与1G8针对相同或相近的表位。为利用单克隆抗体建立检测BoIFN-γ抗原捕获ELISA方法诊断牛疫病,及重组BoIFN-γ的亲和层析纯化提供了物质材料,为深入研究BoIFN-γ单抗在牛临床免疫学方面的应用奠定了基础。
Interferon-gamma, characteristically produced by T lymphocytes and natural killer cells which had been stimulated by antigen and mitogen and plays a critical role in anti-virus, anti-tumor and immune regulation. A lot of studies and applications had been developed with BoIFN-γin internal and abroad, it all needs a convenient and clipping test facility whether in the studies on immunocyte functions and cellular immunologic response or in determination about IFN-γlevel under morbid state. Therefore, the monoclone antibody with BoIFN-γhad been wide applied in the studies of immunologic mechanism and immunologic fuction test.
4~6 weeks BALB/c mice were immunized three times with purified rHis-BoIFN-γprotein, then fusioned mouse myeloma cells and spleen cells of BALB/c mice. Twelve hybridoma clones secreting antibody against bovine interferon-gamma strains against rBoIFN-γwere obtained by screening with the indirect ELISA and 3~5 times subcloning. Twelve MAbs were blonging to IgG isotype,in which five McAbs were blonging to IgG1 isotype, three McAbs were blonging to IgG2a isotype, four McAbs were blonging to IgG isotype. The results revealed that the twelve hybridoma cells strains could secret stably in vitro the McAbs against bovine IFN-γ. Western blot and indirect ELISA showed twelve mAbs reacted to rBoIFN-γ. They also showed strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BoIFN-γ, and suggesting that twelve McAbs were rBoIFN-γspecific monoclonal antibodies. ELISA showed that , 3C2 and 1H4、1G6 and 1G4 were for the different one;1A10、1B2、2B9、3C4 ,1C1 and 1G6, 1G4 and 3C2, 1A10 and 1G8 were for the same epitope. The study were established the groundwork in Immunology and disease diagnosis of bovine IFN-γMcAb.
本研究用原核表达的重组牛IFN-γ(rHis-BoIFN-γ)免疫6周龄BALB/c雌性小鼠,免疫3次后取其脾细胞和骨髓瘤细胞SP2/0进行融合。用建立的间接ELISA方法筛选阳性杂交瘤细胞,经3~5次亚克隆,最终获得12株能稳定分泌抗体的阳性杂交瘤细胞株。亚类鉴定结果显示12株杂交瘤细胞均为IgG,其中5株为IgG1,3株为IgG2a,4株为IgG2b。12株杂交瘤细胞在体外具有稳定分泌抗BoIFN-γ单克隆抗体的能力。Western-blot及间接ELISA证明12株单抗均可特异性的结合rBoIFN-γ蛋白。通过间接免疫荧光显示,12株单抗均与真核细胞BHK21表达的rBoIFN-γ反应产生荧光反应,证明了单抗能够与真核细胞产生的BoIFN-γ反应,证实了单抗的特异性。ELISA叠加试验表明,3C2与1H4、1G6与1G4针对不同表位,1A10、1B2、2B9、3C4 4株和1C1与1G6、1G4与3C2、1A10与1G8针对相同或相近的表位。为利用单克隆抗体建立检测BoIFN-γ抗原捕获ELISA方法诊断牛疫病,及重组BoIFN-γ的亲和层析纯化提供了物质材料,为深入研究BoIFN-γ单抗在牛临床免疫学方面的应用奠定了基础。
Interferon-gamma, characteristically produced by T lymphocytes and natural killer cells which had been stimulated by antigen and mitogen and plays a critical role in anti-virus, anti-tumor and immune regulation. A lot of studies and applications had been developed with BoIFN-γin internal and abroad, it all needs a convenient and clipping test facility whether in the studies on immunocyte functions and cellular immunologic response or in determination about IFN-γlevel under morbid state. Therefore, the monoclone antibody with BoIFN-γhad been wide applied in the studies of immunologic mechanism and immunologic fuction test.
4~6 weeks BALB/c mice were immunized three times with purified rHis-BoIFN-γprotein, then fusioned mouse myeloma cells and spleen cells of BALB/c mice. Twelve hybridoma clones secreting antibody against bovine interferon-gamma strains against rBoIFN-γwere obtained by screening with the indirect ELISA and 3~5 times subcloning. Twelve MAbs were blonging to IgG isotype,in which five McAbs were blonging to IgG1 isotype, three McAbs were blonging to IgG2a isotype, four McAbs were blonging to IgG isotype. The results revealed that the twelve hybridoma cells strains could secret stably in vitro the McAbs against bovine IFN-γ. Western blot and indirect ELISA showed twelve mAbs reacted to rBoIFN-γ. They also showed strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BoIFN-γ, and suggesting that twelve McAbs were rBoIFN-γspecific monoclonal antibodies. ELISA showed that , 3C2 and 1H4、1G6 and 1G4 were for the different one;1A10、1B2、2B9、3C4 ,1C1 and 1G6, 1G4 and 3C2, 1A10 and 1G8 were for the same epitope. The study were established the groundwork in Immunology and disease diagnosis of bovine IFN-γMcAb.
引文
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额尔敦,方建华,郭美香,等. 1998.人IFN-γ免疫PCR检测方法的建立及其敏感性的比较.内蒙古大学学报(自然科学版).2(1): 91~95.
黄诚. 1997.细胞因子对下丘脑-垂体-性腺轴的调控.国外医学·内分泌学分册. 17, 16~19.
金伯泉. 2002.细胞和分子免疫学实验技术.第四军医大学出版社. 16.
金红岩. 2008.鹅γ-干扰素单克隆抗体的制备与鉴定.东北农业大学硕士学位论文.
刘思莹. 2009.抗牛γ-干扰素单克隆抗体制备和鉴定.
李洪涛,马波,王君伟,等. 2007.鹅γ-干扰素成熟蛋白的表达及其生物学活性研究.中国农业科学. 40(1): 190~195.
李瑞芳. 2006.抗BoIFN-gamma特异性单克隆抗体的研制及其特性.中国人兽共患病病学报. 22(8): 755~758.
李川,谭亚娣,陈颖钰,等. 2007.牛IFN-γ原核表达、单克隆抗体制备及其ELISA检测方法的建立.生物工程学报. 23(01): 40~45.
马良. 1995.干扰素与寄生虫感染.重庆医科大学学报. 20(3): 241~244.
秦立廷. 2006.牛、鸡和猪γ-干扰素的克隆表达与单克隆抗体的研制.山东农业大学硕士学位论文.
沈关心,周汝麟.现代免疫学技术.第2版,湖北:湖北科学技术出版社, 2002, 377~379.
田美娜,李永亮,卢增军,等.口蹄疫病毒非结构蛋白3D蛋白单克隆抗体的制备及鉴定.细胞与分子免疫学杂志, 2009, 25(7): 625~627.
屠宗青,沈幼美,童葵塘. 2001.用国产单克隆抗体纯化重组干扰素α1b的研究.微生物学免疫学进展. 29(2): 53~56.
吴美英,艾艳萍,任韧,等. 2002.重组人α-干扰素单克隆抗体的研制及应用.中华实验和临床病毒学杂志. 16(3): 261~263.
孙卫民,王惠琴. 1999.细胞因子研究方法学.人民卫生出版社.
许辉,安献禄,刘雪松,等. 1992.用单克隆抗体亲和层析法纯化重组人肿瘤坏死因子α.免疫学杂志. 8(2): 87~89.
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