日本血吸虫14-3-3蛋白的表达与诊断价值研究
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摘要
制备可溶性表达的重组日本血吸虫信号传导蛋白14-3-3(r Sj14-3-3)并优化表达条件,分析其免疫学特性和免疫诊断价值。成功构建了BL21/p ET30a-r Sj14-3-3表达菌株,最佳的表达体系为细菌生长至OD600为0.6~1.0时加入IPTG至最佳终浓度0.1 mmol/L,28℃诱导16 h。上清液中可溶性的r Sj14-3-3蛋白纯化后的浓度为2.804 mg/m L。以r Sj14-3-3为抗原,间接ELISA检测显示,48份急性与35份慢性血吸虫病患者血清的抗体阳性率分别为95.6%和92.0%;与27份华支睾吸虫和15份钩虫患者血清的交叉反应率分别为7.4%和6.7%;与30份健康人血清假阳性反应率为0%。抗r Sj14-3-3兔血清及所制备的多抗可被纯化出的r Sj14-3-3蛋白及血吸虫成虫抗原(AWA)和排泄分泌物(ESA)中的天然Sj14-3-3分子的反应,且多抗效价达1:1 280 000。本研究成功表达并纯化出r Sj14-3-3蛋白,优化了其诱导表达条件,且此蛋白具有较高的抗原特异性。
The aim of this study was to obtain the soluble recombinant r Sj14-3-3 protein of Schistosoma japonicum and optimized expression condition, then investigate its immunological features and value in immunological diagnosis. Recombinant plasmid p ET30 a/r Sj14-3-3 was constructed successfully. The best expression system of r Sj14-3-3 protein: IPTG was added to a final concentration 0.1 mmol/L when the OD600 of the culture reached 0.6~1.0 and then incubated at 28℃ for 16 h. The concentration of soluble r Sj14-3-3 protein in the supernatant was about 2.804 mg/m L after purification. With RSj14-3-3 as an antigen, indirect ELISA detection showed the positive rate was 95.6% and 92.0% in the serum of 48 patients with acute schistosomiasis and35 patients with chronic schistosomiasis, respectively. The cross reaction rates were 7.4% and 6.7% for the serum of 27 patients with Clonorchis sinensis and 15 nematodes respectively, and false-positive-rate was 0% with 30 healthy people. Rabbit antiserum and polyclonal antibody against r Sj14-3-3 could be recognized by purified r Sj14-3-3, natural Sj14-3-3 in the adult worm antigens(AWAs) and excreted secreted antigens(ESAs). The prepared polyclonal antibody had a high titer(1: 1 280 000). In this research, using the prokaryotic expression system, Sj14-3-3 protein was expressed and purified successfully and it had a high antigen specificity.
The aim of this study was to obtain the soluble recombinant r Sj14-3-3 protein of Schistosoma japonicum and optimized expression condition, then investigate its immunological features and value in immunological diagnosis. Recombinant plasmid p ET30 a/r Sj14-3-3 was constructed successfully. The best expression system of r Sj14-3-3 protein: IPTG was added to a final concentration 0.1 mmol/L when the OD600 of the culture reached 0.6~1.0 and then incubated at 28℃ for 16 h. The concentration of soluble r Sj14-3-3 protein in the supernatant was about 2.804 mg/m L after purification. With RSj14-3-3 as an antigen, indirect ELISA detection showed the positive rate was 95.6% and 92.0% in the serum of 48 patients with acute schistosomiasis and35 patients with chronic schistosomiasis, respectively. The cross reaction rates were 7.4% and 6.7% for the serum of 27 patients with Clonorchis sinensis and 15 nematodes respectively, and false-positive-rate was 0% with 30 healthy people. Rabbit antiserum and polyclonal antibody against r Sj14-3-3 could be recognized by purified r Sj14-3-3, natural Sj14-3-3 in the adult worm antigens(AWAs) and excreted secreted antigens(ESAs). The prepared polyclonal antibody had a high titer(1: 1 280 000). In this research, using the prokaryotic expression system, Sj14-3-3 protein was expressed and purified successfully and it had a high antigen specificity.
引文
Alimu R.Y.L.G.L.,Mao X.F.,and Liu Z.Y.,2013,Expression optimization and ch aracterization of Tenebrio molitor antimicrobiol peptides Tm AMP1m in Escherichia coli,Shengwugongcheng Xuebao(Chinese Journal of Biotechnology),29(6):836-847(热依汗古丽·阿里木,毛新芳,刘忠渊,2013,黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析,生物工程学报,29(6):836-847)
Gray D.J.,Ross A.G.,and Li Y.S.,2011,Diagnosis and management of schistosomiasis,BMJ(Clinical research ed.),342:d2651
He B.F.,Mi L.,and Chen W.H.,2012,Research advances in mechanism of protein secretion and secretory expression of recombinant protein in E.coli,Shipin Yu Shengwujishu Xuebao(Journal of Food Science and Biotechnology),31(6):561-569(何冰芳,米兰,陈文华,2012,大肠杆菌蛋白质分泌机理及其重组蛋白分泌表达新进展,食品与生物技术学报,31(6):561-569)
Hua W.Q.,Xu Y.L.,Yu C.X.,Dai J.R.,He W.,and Cao G.Q.,2005,Analysis of early diagnostic fraction antigens of cercariae,adult worms and eggs of Schistosoma japonicum,Zhongguo Xuexichongbing Fangzhi Zazhi(Chinese Journal of Schistosomiasis Control),17(5):351-355(华万全,许永良,余传信,戴建荣,何伟,曹国群,2005,日本血吸虫尾蚴成虫及虫卵的早期诊断组分抗原分析,中国血吸虫病防治杂志,17(5):351-355)
Isnard A.,and Chevillard C.,2008,Recent advances in the char acterization of genetic factors involved in human susceptibility to infection by schistosomiasis,Current Genomics,9(5):290-300
Jan C.,and Alfons V.G.,2011,Schistosomiasis in travelers and immigrants,Travel Medicine and Infectious Disease,9(1):6-24
Ju X.,Chen C.L.,Liu X.,Zhang T.,Wu S.Q.,and Zhang X.J.,2015,Prokaryotic expression and immunoprotection potential of E.coli outer membrane protein Omp C,Shengmingkexue Yanjiu(Life Science Research),19(2):131-136(俱雄,陈春琳,刘祥,张涛,吴三桥,张晓娟,2015,大肠埃希菌外膜蛋白Omp C的原核表达与免疫保护作用研究,生命科学研究,19(2):131-136)
Li D.F.,Shen J.L.,Zu Y.,and Liu Q.Z.,2003,The purification and the immunoproperties analysis of r Sj14-3-3 protein,Bengbu Yixueyuan Xuebao(Journal of Bengbu Medicine College),28(1):1-3(李德发,沈继龙,祖莹,刘庆中,2003,重组日本血吸虫14-3-3蛋白的纯化和免疫特性分析,蚌埠医学院学报,28(1):1-3)
Li M.,2007,Evaluation of recombinant signaling protein 14-3-3of Schistosoma japonicum on immunodiagnosis of schistosomiasis and the construction of S.japonicum eggs c DNA library,Thesis for M.S.,Anhui Medical University,Supervi sor:Shen J.L.,pp.22-28(李敏,2007,日本血吸虫信号蛋白14-3-3的免疫诊断及虫卵c DNA文库的构建,硕士学位论文,安徽医科大学,导师:沈继龙,pp.22-28)
Liu Q.Z.,2003,Studies on the immunolocalization and protective immunity of the signaling protein 14-3-3 of Schistoma japonicum(Chinese mainland strain)and its diagnostic value,Thesis for M.S.,Anhui Medical University,Supervisor:Shen J.L.,pp.76-84(刘庆中,2003,日本血吸虫(中国大陆株)信号蛋白14-3-3免疫定位、免疫保护作用及其诊断价值的研究,硕士学位论文,安徽医科大学,导师:沈继龙,pp.76-84)
Liu Q.Z.,Shen J.L.,and Wang X.L.,2003,Immunolo calization of the signaling protein 14-3-3 of Schistosoma japonicum,Zhongguo Jishengchongxue Yu Jishengchongbing Zazhi(Chinese Journal of Parasitology and Parasitic Diseases),21(6):330-332(刘庆中,沈继龙,汪学龙,2003,日本血吸虫信号蛋白14-3-3的虫体免疫定位,中国寄生虫学与寄生虫病杂志,21(6):330-332)
Lu Y.,Xu B.,Ju C.,Mo X.J.,Chen S.B.,F Z.,Wang X.N.,and Hu W.,2012,Immunoscreening of Schistosoma japonicum egg c DNA library and identification of positive clones,Zhongguo Jishengchongxue Yu Jishengchongbing Zazhi(Chinese Journal of Parasitology and Parasitic Diseases),30(2):109-115(卢艳,徐斌,鞠川,莫筱瑾,陈绅波,冯正,王小宁,胡薇,2012,日本血吸虫虫卵c DNA文库的免疫筛选和阳性克隆的鉴定,中国寄生虫学与寄生虫病杂志,30(2):109-115)
Vendrame C.M.,Carvalho M.D.,Yamamoto C.R.,Nakhle M.C.,Carvalho S.A.,and Chieffi P.P.,2001,Evaluation of anti-Schistosoma mansoni Ig G antibodies in patients with chronic sch istos omiasis mansoni before and after specific treatment,Revista do Instituto de Medicina Tropical de S觔o Paulo,43(3):153-159
Wang Z.S.,Xiang Q.J.,Wang H.Y.,and Zhang Y.Z.,2010,Cloning and optimizing expression of a periplasmic solute-binding gene gsi B from Escherichia coli,Yichuan(Hereditas),32(5):505-511(王中山,向泉桔,王海燕,张义正,2010,大肠杆菌细胞周质底物结合蛋白gsi B基因的克隆及其表达条件的优化,遗传,32(5):505-511)
Wu G.L.,2002,A historical perspective on the immunodiagnosis of schistosomiasis in China,Acta Tropica,82(2):193-198
Yin X.R.,Xu C.X.,Xie S.Y.,Qian C.Y.,Song L.J.,Wang J.,Zhang W.,and Xu Y.L.,2011,Preparation and characteristics of Schistosoma japonicum signaling protein 14-3-3,Zhongguo Xuexichongbing Fangzhizazhi(Chinese Journal of Schistosomiasis Control),23(2):168-172(殷旭仁,余传信,谢曙英,钱春艳,宋丽君,王玠,张伟,许永良,2011,日本血吸虫信号传导蛋白14-3-3的制备及其特性分析,中国血吸虫病防治杂志,23(2):168-172)
Zhang S.J.,Di J.J.,and Zhang G.W.,2016,Construction of the prokaryotic expression system of yfi F gene in E.coli,optimization of its induced expression condition and purification of the protein,Shengwu Jishu(Biotechnology),26(3):229-233(张树军,狄建军,张国文,2016,大肠杆菌yfi F基因原核表达系统构建、表达条件优化及蛋白纯化,生物技术,26(3):229-233)
Zhang Y.,Bickle Q.D.,and Taylor M.G.,2000,Cloning of Schistosoma japonicum 114-3-3 epsilon(Sj14-3-3 epsilon),a new member of the 14-3-3 family of proteins from schistosomes,International Journal for Parasitology,30(9):991-994
Zhang Y.,Taylor M.G.,Mc Crossan M.V.,and Bickle Q.D.,1999,Molecular cloning and characterization of a novel Schistosoma japonicum“irradiated vaccine-specific”anti gene,Sj14-3-3,Molecular and biochemical parasitology,10(1):25-34
Zhou L.,2011,Establishment of an assay for early detection of Schistosoma species and production of transgenic mice with IL-22 specific expression,Dissertation for Ph.D.,Wuhan U-niversity,Supervisor:Wang Y.F.,pp.23-25(周立,2011,血吸虫病早期诊断方法和白介素22转基因小鼠模型的建立,博士学位论文,武汉大学,导师:王业富,pp.23-25)
Gray D.J.,Ross A.G.,and Li Y.S.,2011,Diagnosis and management of schistosomiasis,BMJ(Clinical research ed.),342:d2651
He B.F.,Mi L.,and Chen W.H.,2012,Research advances in mechanism of protein secretion and secretory expression of recombinant protein in E.coli,Shipin Yu Shengwujishu Xuebao(Journal of Food Science and Biotechnology),31(6):561-569(何冰芳,米兰,陈文华,2012,大肠杆菌蛋白质分泌机理及其重组蛋白分泌表达新进展,食品与生物技术学报,31(6):561-569)
Hua W.Q.,Xu Y.L.,Yu C.X.,Dai J.R.,He W.,and Cao G.Q.,2005,Analysis of early diagnostic fraction antigens of cercariae,adult worms and eggs of Schistosoma japonicum,Zhongguo Xuexichongbing Fangzhi Zazhi(Chinese Journal of Schistosomiasis Control),17(5):351-355(华万全,许永良,余传信,戴建荣,何伟,曹国群,2005,日本血吸虫尾蚴成虫及虫卵的早期诊断组分抗原分析,中国血吸虫病防治杂志,17(5):351-355)
Isnard A.,and Chevillard C.,2008,Recent advances in the char acterization of genetic factors involved in human susceptibility to infection by schistosomiasis,Current Genomics,9(5):290-300
Jan C.,and Alfons V.G.,2011,Schistosomiasis in travelers and immigrants,Travel Medicine and Infectious Disease,9(1):6-24
Ju X.,Chen C.L.,Liu X.,Zhang T.,Wu S.Q.,and Zhang X.J.,2015,Prokaryotic expression and immunoprotection potential of E.coli outer membrane protein Omp C,Shengmingkexue Yanjiu(Life Science Research),19(2):131-136(俱雄,陈春琳,刘祥,张涛,吴三桥,张晓娟,2015,大肠埃希菌外膜蛋白Omp C的原核表达与免疫保护作用研究,生命科学研究,19(2):131-136)
Li D.F.,Shen J.L.,Zu Y.,and Liu Q.Z.,2003,The purification and the immunoproperties analysis of r Sj14-3-3 protein,Bengbu Yixueyuan Xuebao(Journal of Bengbu Medicine College),28(1):1-3(李德发,沈继龙,祖莹,刘庆中,2003,重组日本血吸虫14-3-3蛋白的纯化和免疫特性分析,蚌埠医学院学报,28(1):1-3)
Li M.,2007,Evaluation of recombinant signaling protein 14-3-3of Schistosoma japonicum on immunodiagnosis of schistosomiasis and the construction of S.japonicum eggs c DNA library,Thesis for M.S.,Anhui Medical University,Supervi sor:Shen J.L.,pp.22-28(李敏,2007,日本血吸虫信号蛋白14-3-3的免疫诊断及虫卵c DNA文库的构建,硕士学位论文,安徽医科大学,导师:沈继龙,pp.22-28)
Liu Q.Z.,2003,Studies on the immunolocalization and protective immunity of the signaling protein 14-3-3 of Schistoma japonicum(Chinese mainland strain)and its diagnostic value,Thesis for M.S.,Anhui Medical University,Supervisor:Shen J.L.,pp.76-84(刘庆中,2003,日本血吸虫(中国大陆株)信号蛋白14-3-3免疫定位、免疫保护作用及其诊断价值的研究,硕士学位论文,安徽医科大学,导师:沈继龙,pp.76-84)
Liu Q.Z.,Shen J.L.,and Wang X.L.,2003,Immunolo calization of the signaling protein 14-3-3 of Schistosoma japonicum,Zhongguo Jishengchongxue Yu Jishengchongbing Zazhi(Chinese Journal of Parasitology and Parasitic Diseases),21(6):330-332(刘庆中,沈继龙,汪学龙,2003,日本血吸虫信号蛋白14-3-3的虫体免疫定位,中国寄生虫学与寄生虫病杂志,21(6):330-332)
Lu Y.,Xu B.,Ju C.,Mo X.J.,Chen S.B.,F Z.,Wang X.N.,and Hu W.,2012,Immunoscreening of Schistosoma japonicum egg c DNA library and identification of positive clones,Zhongguo Jishengchongxue Yu Jishengchongbing Zazhi(Chinese Journal of Parasitology and Parasitic Diseases),30(2):109-115(卢艳,徐斌,鞠川,莫筱瑾,陈绅波,冯正,王小宁,胡薇,2012,日本血吸虫虫卵c DNA文库的免疫筛选和阳性克隆的鉴定,中国寄生虫学与寄生虫病杂志,30(2):109-115)
Vendrame C.M.,Carvalho M.D.,Yamamoto C.R.,Nakhle M.C.,Carvalho S.A.,and Chieffi P.P.,2001,Evaluation of anti-Schistosoma mansoni Ig G antibodies in patients with chronic sch istos omiasis mansoni before and after specific treatment,Revista do Instituto de Medicina Tropical de S觔o Paulo,43(3):153-159
Wang Z.S.,Xiang Q.J.,Wang H.Y.,and Zhang Y.Z.,2010,Cloning and optimizing expression of a periplasmic solute-binding gene gsi B from Escherichia coli,Yichuan(Hereditas),32(5):505-511(王中山,向泉桔,王海燕,张义正,2010,大肠杆菌细胞周质底物结合蛋白gsi B基因的克隆及其表达条件的优化,遗传,32(5):505-511)
Wu G.L.,2002,A historical perspective on the immunodiagnosis of schistosomiasis in China,Acta Tropica,82(2):193-198
Yin X.R.,Xu C.X.,Xie S.Y.,Qian C.Y.,Song L.J.,Wang J.,Zhang W.,and Xu Y.L.,2011,Preparation and characteristics of Schistosoma japonicum signaling protein 14-3-3,Zhongguo Xuexichongbing Fangzhizazhi(Chinese Journal of Schistosomiasis Control),23(2):168-172(殷旭仁,余传信,谢曙英,钱春艳,宋丽君,王玠,张伟,许永良,2011,日本血吸虫信号传导蛋白14-3-3的制备及其特性分析,中国血吸虫病防治杂志,23(2):168-172)
Zhang S.J.,Di J.J.,and Zhang G.W.,2016,Construction of the prokaryotic expression system of yfi F gene in E.coli,optimization of its induced expression condition and purification of the protein,Shengwu Jishu(Biotechnology),26(3):229-233(张树军,狄建军,张国文,2016,大肠杆菌yfi F基因原核表达系统构建、表达条件优化及蛋白纯化,生物技术,26(3):229-233)
Zhang Y.,Bickle Q.D.,and Taylor M.G.,2000,Cloning of Schistosoma japonicum 114-3-3 epsilon(Sj14-3-3 epsilon),a new member of the 14-3-3 family of proteins from schistosomes,International Journal for Parasitology,30(9):991-994
Zhang Y.,Taylor M.G.,Mc Crossan M.V.,and Bickle Q.D.,1999,Molecular cloning and characterization of a novel Schistosoma japonicum“irradiated vaccine-specific”anti gene,Sj14-3-3,Molecular and biochemical parasitology,10(1):25-34
Zhou L.,2011,Establishment of an assay for early detection of Schistosoma species and production of transgenic mice with IL-22 specific expression,Dissertation for Ph.D.,Wuhan U-niversity,Supervisor:Wang Y.F.,pp.23-25(周立,2011,血吸虫病早期诊断方法和白介素22转基因小鼠模型的建立,博士学位论文,武汉大学,导师:王业富,pp.23-25)