新孢子虫致密颗粒7和鼠白介素18融合蛋白的表达及鉴定
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摘要
目的获取新孢子虫致密颗粒7和鼠白介素18(NcGRA7-MouseIL-18)重组基因,表达NcGRA7-MouseIL-18融合蛋白并进行纯化。方法以新孢子虫的cDNA和鼠巨噬细胞的cDNA为模板,采用PCR技术及无缝克隆技术(In-Fusion Cloning)扩增NcGRA7-MouseIL-18基因片段,构建pET-32a-NcGRA7-MouseIL-18重组表达质粒,转入E.coli BL21(DE3)感受态细胞中,诱导表达目的蛋白NcGRA7-MouseIL-18,进行SDS-PAGE和Western blot分析;采用Ni-NTA亲和层析柱纯化NcGRA7-MouseIL-18蛋白,并纯化蛋白进行Western blot鉴定。结果酶切和基因测序鉴定质粒pET-32a-NcGRA7-MouseIL-18构建正确,表达带His标签的相对分子质量约70×10~3的可溶性NcGRA7-MouseIL-18蛋白,与预期值相符;未纯化和纯化的融合蛋白均可被鼠抗His单抗识别。结论成功构建了pET-32a-NcGRA7-MouseIL-18重组质粒,表达的NcGRA7-MouseIL-18融合蛋白具有反应原性。
Objective To express the NcGRA7-mouse IL-18 fusion gene in E.coli BL21(DE3) and to purify the fusion protein. Methods The NcGRA7-mouse IL-18 fusion gene was amplified with PCR and In-Fusion Cloning using the cDNA of Neospora caninum and mouse macrophages as respective templates. The pET-32 a-NcGRA7-mouse IL-18 plasmid was transformed into E.coli BL21(DE3) competent cells. The expressed protein NcGRA7-mouse IL-18 was identified using SDS-PAGE and Western blotting. Results The plasmid pET-32 a-NcGRA7-mouse IL-18 was verified as correct according to enzyme digestion and DNA sequencing. The expressed protein was a soluble protein with a relative molecular mass of about 70×10~3 with an His tag. The size of the protein was consistent with the expected value. The expressed protein specifically bound to anti-mouse His antibody, indicating that the fusion protein had good reactivity. Conclusion An NcGRA7-mouse IL-18 recombinant plasmid was successfully constructed, and the fusion protein had good reactivity.
Objective To express the NcGRA7-mouse IL-18 fusion gene in E.coli BL21(DE3) and to purify the fusion protein. Methods The NcGRA7-mouse IL-18 fusion gene was amplified with PCR and In-Fusion Cloning using the cDNA of Neospora caninum and mouse macrophages as respective templates. The pET-32 a-NcGRA7-mouse IL-18 plasmid was transformed into E.coli BL21(DE3) competent cells. The expressed protein NcGRA7-mouse IL-18 was identified using SDS-PAGE and Western blotting. Results The plasmid pET-32 a-NcGRA7-mouse IL-18 was verified as correct according to enzyme digestion and DNA sequencing. The expressed protein was a soluble protein with a relative molecular mass of about 70×10~3 with an His tag. The size of the protein was consistent with the expected value. The expressed protein specifically bound to anti-mouse His antibody, indicating that the fusion protein had good reactivity. Conclusion An NcGRA7-mouse IL-18 recombinant plasmid was successfully constructed, and the fusion protein had good reactivity.
引文
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[13] Lu Q, Xing S, Gong P, et al. A 78 kDa host cell invasion protein of Neospora caninum as a potential vaccine candidate[J]. Exp Parasitol, 2015(148): 56-65.
[14] Jenkins M, Paker C, Tuo W, et al. Inclusion of CpG adjuvant with plasmid DNA coding for NcGRA7 improves protection against congenital neosporosis[J]. Infect Immun, 2004, 72(3): 1817.
[15] 黄艺华. 鸡堆型艾美耳球虫Rhomboid增强型核酸疫苗构建及免疫保护作用[D]. 哈尔滨: 东北农业大学, 2016.
[16] 高佩. 鸭IFNα-IFNγ、IFNα-IL18基因的融合表达及其生物学活性研究[D]. 广州: 华南农业大学, 2016.
[17] Wan-chi TB, Joan RP, Matthias L, et al. IL-18 Inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms[J]. Plos One, 2011, 6(9): e24241.
[18] 徐玢, 马小彤, 董成亚, 等. 小鼠IL-18基因修饰瘤苗的抗白血病作用研究[J]. 现代免疫学, 2007, 27(4): 274-8.
[19] Takashima Y, Takasu M, Yanagimoto I, et al. Prevalence and dynamics of antibodies against NcSAG1 and NcGRA7 antigens of Neospora caninum in cattle during the gestationperiod[J]. J Vet Med Sci, 2013, 75(11): 1413-8.
[20] 金春梅, 许应天, 高旭, 等. 新孢子虫NcGRA2蛋白多克隆抗体的制备及其亚细胞定位[J]. 中国病原生物学杂志, 2018, 13(12): 1372-4.
[21] Hiasa J, Kohara J, Nishimura M, et al. ELISAs based on rNcGRA7 and rNcSAG1 antigens as an indicator of Neospora caninum activation[J]. Vet Parasitol, 2012, 187(3): 379-85.
[22] Nishikawa Y, Zhang H, Ikehara Y, et al. Immunization with oligomannose-coated liposome-entrapped dense granule protein 7 protects dams and offspring from Neospora caninum infection in mice[J]. Clin Vaccine Immunol, 2009, 16(6): 792-7.
[23] Ferreirinha P, Correia A, Teixeira-Coelho M, et al. Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection [J]. Vaccine, 2016, 34(50): 6250-8.
[24] 门静涛, 刘全, 商利民, 等. IL-18增强日本血吸虫DNA疫苗Sj23的免疫保护效果(英文) [J]. 中国兽医学报, 2009, 29(05): 610-4.
[25] Wei F, Liu Q, Zhai Y, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine [J]. Acta Tropica, 2009, 111(3): 284-8.
[26] Wei F, Liu Q, Gao S, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26kDa GST plasmid DNA vaccine in mice [J]. Vaccine, 2008, 26(33): 4145-9.
[27] 包剑铭, 郭小帆, 赵雪飞, 等. 大肠杆菌碱性磷酸酶在E.coli BL21(DE3)中的胞内可溶性表达[J]. 中国生化药物杂志, 2014, 34(5): 164-6.
[28] 金春梅, 于龙政, 张守发. 新孢子虫致密颗粒蛋白截短型NcGRA7t的体外表达及免疫活性检测[J]. 畜牧与兽医, 2012, 44(12): 57-9.
[29] 舒黛廉, 王珏. 猪α干扰素基因在pET32a表达载体中的表达和鉴定[J]. 中国兽医杂志, 2013, 49(7): 32-5.
[30] 陈磊, 刘成倩, 李红, 等. pET-32a载体介导的生长抑制素基因在大肠杆菌中表达[J]. 上海农业学报, 2016, 32(1): 15-8.
[31] 张婷婷. 丹参C4H基因原核表达载体的构建及重组蛋白的纯化[D]. 杨凌: 西北农林科技大学, 2011.
[32] 智庆文, 苗爱玲. TAT-hEGF融合蛋白在E.coli BL21(DE3)中高效自我表达[J]. 生物技术通报, 2010(7): 89-91, 100.
[2] Dubey JP, Carpenter JL, Speer CA, et al. Newly recognized fatal protozoan disease of dogs [J]. J Am Vet Med Assoc, 1988, 192(9): 1269-85.
[3] 张西臣, 李建华. 动物寄生虫病学[M]. 4版. 成都: 四川出版集团, 2005.
[4] Abdelbaky HH, Fereig RM, Nishikaw AY. Identification of the antigenic region of Neospora caninum dense granule protein 7 using ELISA [J]. Parasitol Int, 2018, 67(6): 675.
[5] 翟涛, 宫鹏涛, 张西臣, 等. 新孢子虫感染小鼠巨噬细胞IFN-γ、TNF-α和IL-6分泌的变化[J]. 中国病原生物学杂志, 2017, 12(4): 350-2, 358.
[6] 康晓冬, 高海慧, 脱征军, 等. 宁夏地区奶牛新孢子虫病的流行病学调查[J]. 中国畜牧兽医文摘, 2017, 33(09): 111-9.
[7] Hosseininejad M, Mahzounieh M, Shams NE. Neospora caninum suspects as one of the most important causes of abortion in large dairy farms in Isfahan, Iran[J]. Iran J Parasitol, 2017, 12(3): 408-12.
[8] Yang Y, Zhang Q, Kong Y, et al. Low prevalence of Neospora caninum and Toxoplasma gondii antibodies in dogs in Jilin, Henan and Anhui Provinces of the People's Republic of China[J]. Bmc Vet Res, 2014, 10(1): 1-6.
[9] 柴方红, 孙兴忠, 李航, 等. 吉林省西部地区牛新孢子虫病流行病学调查[J]. 延边大学农学学报, 2018, 40(2): 82-5.
[10] 李坤, 高建峰, 韩照清, 等. 西藏部分地区黄牛新孢子虫流行病学调查[J]. 中国奶牛, 2014(14): 57-8.
[11] 贾立军, 张守发, 李男礼. 牛源犬新孢子虫NcGRA7基因的克隆及原核表达分析[J]. 中国预防兽医学报, 2013, 35(9): 773-5.
[12] Liddell S, Parker C, Vinyard B, et al. Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum[J]. J Parasitol, 2003, 89(3): 496-500.
[13] Lu Q, Xing S, Gong P, et al. A 78 kDa host cell invasion protein of Neospora caninum as a potential vaccine candidate[J]. Exp Parasitol, 2015(148): 56-65.
[14] Jenkins M, Paker C, Tuo W, et al. Inclusion of CpG adjuvant with plasmid DNA coding for NcGRA7 improves protection against congenital neosporosis[J]. Infect Immun, 2004, 72(3): 1817.
[15] 黄艺华. 鸡堆型艾美耳球虫Rhomboid增强型核酸疫苗构建及免疫保护作用[D]. 哈尔滨: 东北农业大学, 2016.
[16] 高佩. 鸭IFNα-IFNγ、IFNα-IL18基因的融合表达及其生物学活性研究[D]. 广州: 华南农业大学, 2016.
[17] Wan-chi TB, Joan RP, Matthias L, et al. IL-18 Inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms[J]. Plos One, 2011, 6(9): e24241.
[18] 徐玢, 马小彤, 董成亚, 等. 小鼠IL-18基因修饰瘤苗的抗白血病作用研究[J]. 现代免疫学, 2007, 27(4): 274-8.
[19] Takashima Y, Takasu M, Yanagimoto I, et al. Prevalence and dynamics of antibodies against NcSAG1 and NcGRA7 antigens of Neospora caninum in cattle during the gestationperiod[J]. J Vet Med Sci, 2013, 75(11): 1413-8.
[20] 金春梅, 许应天, 高旭, 等. 新孢子虫NcGRA2蛋白多克隆抗体的制备及其亚细胞定位[J]. 中国病原生物学杂志, 2018, 13(12): 1372-4.
[21] Hiasa J, Kohara J, Nishimura M, et al. ELISAs based on rNcGRA7 and rNcSAG1 antigens as an indicator of Neospora caninum activation[J]. Vet Parasitol, 2012, 187(3): 379-85.
[22] Nishikawa Y, Zhang H, Ikehara Y, et al. Immunization with oligomannose-coated liposome-entrapped dense granule protein 7 protects dams and offspring from Neospora caninum infection in mice[J]. Clin Vaccine Immunol, 2009, 16(6): 792-7.
[23] Ferreirinha P, Correia A, Teixeira-Coelho M, et al. Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection [J]. Vaccine, 2016, 34(50): 6250-8.
[24] 门静涛, 刘全, 商利民, 等. IL-18增强日本血吸虫DNA疫苗Sj23的免疫保护效果(英文) [J]. 中国兽医学报, 2009, 29(05): 610-4.
[25] Wei F, Liu Q, Zhai Y, et al. IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine [J]. Acta Tropica, 2009, 111(3): 284-8.
[26] Wei F, Liu Q, Gao S, et al. Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26kDa GST plasmid DNA vaccine in mice [J]. Vaccine, 2008, 26(33): 4145-9.
[27] 包剑铭, 郭小帆, 赵雪飞, 等. 大肠杆菌碱性磷酸酶在E.coli BL21(DE3)中的胞内可溶性表达[J]. 中国生化药物杂志, 2014, 34(5): 164-6.
[28] 金春梅, 于龙政, 张守发. 新孢子虫致密颗粒蛋白截短型NcGRA7t的体外表达及免疫活性检测[J]. 畜牧与兽医, 2012, 44(12): 57-9.
[29] 舒黛廉, 王珏. 猪α干扰素基因在pET32a表达载体中的表达和鉴定[J]. 中国兽医杂志, 2013, 49(7): 32-5.
[30] 陈磊, 刘成倩, 李红, 等. pET-32a载体介导的生长抑制素基因在大肠杆菌中表达[J]. 上海农业学报, 2016, 32(1): 15-8.
[31] 张婷婷. 丹参C4H基因原核表达载体的构建及重组蛋白的纯化[D]. 杨凌: 西北农林科技大学, 2011.
[32] 智庆文, 苗爱玲. TAT-hEGF融合蛋白在E.coli BL21(DE3)中高效自我表达[J]. 生物技术通报, 2010(7): 89-91, 100.