抗牛γ-干扰素单克隆抗体制备和鉴定
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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(ConA、PHA)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、抑制细胞增殖和免疫调节等多种生物学活性的糖蛋白。自Cerretti于1986年首次克隆出乳牛IFN-γ基因后,重组牛IFN-γ(rBoIFN-γ)先后在原核表达系统、真核表达系统、巴斯德毕赤氏酵母系统、牛I型疱疹病毒载体(BHV-1)中成功获得了表达,并对其免疫学和生物学活性进行了研究。国内外关于牛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。
本研究通过常规PCR方法从pMD18-T-BoIFN-γ质粒中扩增出BoIFN-γ成熟蛋白基因,并将其插入原核表达载体pET30a和pGEX-6p-1中,经DNA测序确认后,分别转化入Rosetta(DE3)pLysS中,经IPTG诱导分别表达出约18ku和42ku左右的融合蛋白。以纯化的rHis-BoIFN-γ蛋白作为免疫原,免疫BALB/c小鼠,用纯化的rGST-BoIFN-γ蛋白做为检测抗原,应用淋巴细胞杂交瘤技术,通过间接ELISA方法进行阳性杂交瘤细胞的筛选,共筛选出4株稳定分泌抗rBoIFN-γ的单克隆抗体的细胞株,分别命名为3C9、3D10、2C12、5C4,免疫球蛋白类均为IgM,轻链为κ链;杂交瘤细胞株连续传代15次以上及冻存后复苏,细胞生长状态良好,效价稳定;腹水效价分别为1:51200、1:51200、1:12800、1:6400。Western blot结果表明,4株单抗均可特异性的识别rGST-BoIFN-γ蛋白,而不与GST标签蛋白反应。间接ELISA试验证明,4株单抗只与rBoIFN-γ反应,而不与GST蛋白和rGoIFN-α、rGoIFN-γ、rBoIFN-α等细胞因子发生交叉反应。间接免疫荧光结果表明,4株单抗均与BHK21细胞表达的rBoIFN-γ发生强荧光信号,进一步证明了单抗的特异性。阻断ELISA检测结果表明3D10株单抗能够与天然干扰素发生反应,证实了该单抗具有实际的应用价值。本研究所制备的4株抗BoIFN-γ的单克隆抗体,为进一步研究BoIFN-γ单抗在牛免疫学以及牛疫病诊断中的应用奠定了基础。
Interferon-gamma, characteristically produced by T lymphocytes and natural killer cells which had been stimulated by antigen and mitogen, is a pleiotropic cytokine and plays a critical role in anti-virus, anti-tumor and immune regulation. After first cloned by Cerretti in 1986, BoIFN-γwas successfully expressed in Prokaryotic expression system, eukaryotic expression system, pichia system and bovineⅠtype herpesvirus vector, and its immunology and biologic activity was also studied. 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.
In the study,the mature BoIFN-γ(without signal peptide) was amplified by routine PCR with recombinant plasmid pMD18-T-BoIFN-γas template. Then, the genes were sucloned into pET30a and pGEX-6p-1 respectively. After confirmation by sequencing, the two recombinant plasmids pET30a-BoIFN-γand pGEX-6p-1-BoIFN-γwere transformed into Rosetta(DE3)pLysS. Recombinant bacteria was induced by IPTG, and fusion protein were expressed at anticipated MW 18ku and 42ku band appeared on SDS-PAGE. To prepare monoclonal antibodies against rBoIFN-γ, BALB/c mice were immunized with purified fusion protein rHis-BoIFN-γ. With the purified protein rGST-BoIFN-γas detecting antigen,mAbs against rBoIFN-γwere prepared using lymphocyte hybridoma technique and positive hybridoma clones were screened by indirect ELISA. Four hybridoma cell lines were obtained and named 3C9、3D10、2C12、5C4. The isotypes of four mAbs were IgM, and the light chains areκ; and the ELISA titers of four mAbs hydroperitoneum were 1:51200、1:51200、1:12800 and 1:6400 respectively. Western blot analysis suggested four mAbs could only react with the rGST-BoIFN-γprotein, but not with GST protein. Indirect ELISA showed that four mAbs reacted to rBoIFN-γ, but not reacted to GST protein and cytokine proteins of rGoIFN-α、rGoIFN-γand rBoIFN-α. They also shown strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BoIFN-γ, suggesting that four mAbs were rBoIFN-γspecific monoclonal antibodies. The result of blocking ELISA detection showed McAb 3D10 could reacted with natural IFN-γ, and the McAb has practical value.The study has obtained 4 McAbs which were specific to the BoIFN-γ.
The study were established the groundwork in Immunology and disease diagnosis of bovine IFN-γMcAb.
本研究通过常规PCR方法从pMD18-T-BoIFN-γ质粒中扩增出BoIFN-γ成熟蛋白基因,并将其插入原核表达载体pET30a和pGEX-6p-1中,经DNA测序确认后,分别转化入Rosetta(DE3)pLysS中,经IPTG诱导分别表达出约18ku和42ku左右的融合蛋白。以纯化的rHis-BoIFN-γ蛋白作为免疫原,免疫BALB/c小鼠,用纯化的rGST-BoIFN-γ蛋白做为检测抗原,应用淋巴细胞杂交瘤技术,通过间接ELISA方法进行阳性杂交瘤细胞的筛选,共筛选出4株稳定分泌抗rBoIFN-γ的单克隆抗体的细胞株,分别命名为3C9、3D10、2C12、5C4,免疫球蛋白类均为IgM,轻链为κ链;杂交瘤细胞株连续传代15次以上及冻存后复苏,细胞生长状态良好,效价稳定;腹水效价分别为1:51200、1:51200、1:12800、1:6400。Western blot结果表明,4株单抗均可特异性的识别rGST-BoIFN-γ蛋白,而不与GST标签蛋白反应。间接ELISA试验证明,4株单抗只与rBoIFN-γ反应,而不与GST蛋白和rGoIFN-α、rGoIFN-γ、rBoIFN-α等细胞因子发生交叉反应。间接免疫荧光结果表明,4株单抗均与BHK21细胞表达的rBoIFN-γ发生强荧光信号,进一步证明了单抗的特异性。阻断ELISA检测结果表明3D10株单抗能够与天然干扰素发生反应,证实了该单抗具有实际的应用价值。本研究所制备的4株抗BoIFN-γ的单克隆抗体,为进一步研究BoIFN-γ单抗在牛免疫学以及牛疫病诊断中的应用奠定了基础。
Interferon-gamma, characteristically produced by T lymphocytes and natural killer cells which had been stimulated by antigen and mitogen, is a pleiotropic cytokine and plays a critical role in anti-virus, anti-tumor and immune regulation. After first cloned by Cerretti in 1986, BoIFN-γwas successfully expressed in Prokaryotic expression system, eukaryotic expression system, pichia system and bovineⅠtype herpesvirus vector, and its immunology and biologic activity was also studied. 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.
In the study,the mature BoIFN-γ(without signal peptide) was amplified by routine PCR with recombinant plasmid pMD18-T-BoIFN-γas template. Then, the genes were sucloned into pET30a and pGEX-6p-1 respectively. After confirmation by sequencing, the two recombinant plasmids pET30a-BoIFN-γand pGEX-6p-1-BoIFN-γwere transformed into Rosetta(DE3)pLysS. Recombinant bacteria was induced by IPTG, and fusion protein were expressed at anticipated MW 18ku and 42ku band appeared on SDS-PAGE. To prepare monoclonal antibodies against rBoIFN-γ, BALB/c mice were immunized with purified fusion protein rHis-BoIFN-γ. With the purified protein rGST-BoIFN-γas detecting antigen,mAbs against rBoIFN-γwere prepared using lymphocyte hybridoma technique and positive hybridoma clones were screened by indirect ELISA. Four hybridoma cell lines were obtained and named 3C9、3D10、2C12、5C4. The isotypes of four mAbs were IgM, and the light chains areκ; and the ELISA titers of four mAbs hydroperitoneum were 1:51200、1:51200、1:12800 and 1:6400 respectively. Western blot analysis suggested four mAbs could only react with the rGST-BoIFN-γprotein, but not with GST protein. Indirect ELISA showed that four mAbs reacted to rBoIFN-γ, but not reacted to GST protein and cytokine proteins of rGoIFN-α、rGoIFN-γand rBoIFN-α. They also shown strong reactivity in indirect immunofluorescence test on the BHK21 cell with transient expressed BoIFN-γ, suggesting that four mAbs were rBoIFN-γspecific monoclonal antibodies. The result of blocking ELISA detection showed McAb 3D10 could reacted with natural IFN-γ, and the McAb has practical value.The study has obtained 4 McAbs which were specific to the BoIFN-γ.
The study were established the groundwork in Immunology and disease diagnosis of bovine IFN-γMcAb.
引文
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