应用正交实验法优化大肠杆菌表达CTB-PSMA624-632蛋白的条件
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摘要
通过基因工程方法,将含霍乱毒素B亚单位(CTB)和前列腺特异性膜抗原(PSMA)表位肽的重组质粒pET28a-CTB-PSMA624-632,转化表达宿主菌BL21 (DE3)。CTB蛋白的表达有利于开发粘膜佐剂和霍乱弧菌基因工程疫苗,同时抗原肽疫苗也是肿瘤免疫治疗的理想候选。本研究采用正交实验法,优化条件以增加大肠杆菌中CTB-PSMA624-632蛋白的表达量。选取了5个单因素作为考察因子,分别为:预诱导时间、诱导时间、诱导温度、IPTG浓度、诱导转速。先做了4水平的实验L16(45)。根据极差分析的数据对影响较大的3个因素又做了细调型的3水平实验L9(33)。结果表明,在早期指数生长期的诱导能得到相对高水平的包涵体形式的CTB-PSMA624-632蛋白,并且此蛋白的表达随着诱导温度升高到37℃而逐渐增加;相比于使用正常浓度的IPTG诱导得到的蛋白表达量,用100%μmol/L诱导足以诱导蛋白的表达,这比通常使用的IPTG浓度小10倍;此外,诱导时间5 h与7 h对蛋白的表达量差距甚微,因此选定较短时间的5 h,240 r/min的诱导转速也被认为是能诱导目的蛋白CTB-PSMA624-632具有高表达量的最优条件。
The recombinant plasmid p ET28 a-CTB-PSMA624-632, containing cholera toxin B subunit(CTB) and prostate specific membrane antigen(PSMA) epitope antigen, was transformed and expressed in host strain BL21(DE3) by genetic engineering method. The expression of CTB protein was beneficial to the development of mucosal adjuvant and Vibrio cholera gene engineering vaccine, while antigen peptide vaccine was also an ideal candidate for tumor immunotherapy. Herein, orthogonal experiments were used in this study to optimize the conditions to increase the expression level of CTB-PSMA624-632 protein in Escherichia coli. There were five selected single influencing factors which were post-induction time, induction time, induction temperature, IPTG concentration, and induction speed, respectively. We did a four-level experiment L16(45) at first. According to the range analysis, the three factors that attested most were further studied in the finely adjusting three-level experiment L9(33). The results showed that the relatively high-level CTB-PSMA624-632 protein in the form of inclusion bodies was obtained at the early exponential growth phase, and the expression of this protein increased gradually with the increase of induction temperature to 37℃. Inducting with 100 μmol/L IPTG was sufficient to induce the expression of CTB-PSMA624-632 which was 10 times less than normally used IPTG concentration. In addition, the induction time between 5 h and 7 h showed a little difference on protein expression, accordingly we selected the shorter one.Furthermore, 240 r/min of induction speed was also considered to be optimal to induce the target protein CTBPSMA624-632 with higher expression.
The recombinant plasmid p ET28 a-CTB-PSMA624-632, containing cholera toxin B subunit(CTB) and prostate specific membrane antigen(PSMA) epitope antigen, was transformed and expressed in host strain BL21(DE3) by genetic engineering method. The expression of CTB protein was beneficial to the development of mucosal adjuvant and Vibrio cholera gene engineering vaccine, while antigen peptide vaccine was also an ideal candidate for tumor immunotherapy. Herein, orthogonal experiments were used in this study to optimize the conditions to increase the expression level of CTB-PSMA624-632 protein in Escherichia coli. There were five selected single influencing factors which were post-induction time, induction time, induction temperature, IPTG concentration, and induction speed, respectively. We did a four-level experiment L16(45) at first. According to the range analysis, the three factors that attested most were further studied in the finely adjusting three-level experiment L9(33). The results showed that the relatively high-level CTB-PSMA624-632 protein in the form of inclusion bodies was obtained at the early exponential growth phase, and the expression of this protein increased gradually with the increase of induction temperature to 37℃. Inducting with 100 μmol/L IPTG was sufficient to induce the expression of CTB-PSMA624-632 which was 10 times less than normally used IPTG concentration. In addition, the induction time between 5 h and 7 h showed a little difference on protein expression, accordingly we selected the shorter one.Furthermore, 240 r/min of induction speed was also considered to be optimal to induce the target protein CTBPSMA624-632 with higher expression.
引文
Ding J.Y.,Yi Y.,Lu X.X.,Su Q.D.,Tian R.G.,Qiu F.,and Bi S.L.,2012,Effect of different temperature,time and different concentration of IPTG and strains on HDV Ag expression,Yixue Yanjiu Zazhi(Journal of Medical Research),41(5):31-34(丁军颖,伊瑶,卢学新,苏秋冬,田瑞光,邱丰,毕胜利,2012,不同温度、时间、IPTG和菌种浓度对丁肝抗原蛋白表达量的影响,医学研究杂志,41(5):31-34)
Dvorak P.,Chrast L.,Nikel P.I.,Fedr R.,Soucek K.,Sedlackova M.,Chaloupkova R.,Lorenzo V.,Prokop Z.,and Damborsky J.,2015,Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3)carrying a synthetic metabolic pathway,Microbial Cell Factories.,14(1):201-215
Halin S.,Rudolfsson S.H.,Rooijen N.V.,and Bergh A.,2009,Extratumoral macrophages promote tumor and vascular growth in an orthotopic rat prostate tumor model,Neoplasia.,11(2):177-186
Jin Y.C.,Liu L.,Su X.Y.,Li J.P.,Li H.D.,and Zhou J.H.,2006,Effect of induce concentration,time and temperature of IPTG on the expression of the GST-GnRH/TRS gene,Heilongjiang Xumu Shouyi(Heilongjiang Animal Science and Veterinary Medicine),(8):17-19(金元昌,刘利,苏晓艳,李景鹏,李会东,周建红,2006,IPTG诱导浓度、时间及温度对重组促性腺激素释放激素基因表达的影响,黑龙江畜牧兽医,(8):17-19)
Kawamura Y.I.,Kawashima R.,Shirai Y.,Kato R.,Hamabata T.,Yamamoto M.,Fuukawa K.,Fujihashi K.,Mcghee J.R.,Hayashi H.,and Dohi T.,2003,Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB Translation,Eur.J.Immunol.,33(11):3205-3212
Li W.B.,2013,The research of expression of YAP in prostate cancer and the role of interaction between YAP and AR in CRPC transforming,Thesis for M.S.,Chongqing Medical University,Supervisor:Wang D.L.,pp.12-15(李文宾,2013,Yes相关蛋白在前列腺中的表达及其与AR相互作用在CRPC转化中机制的初步探讨,硕士学位论文,重庆医科大学,导师:王德林,pp.12-15)
Noguchi M.,Sasada T.,and Itoh K.,2013,Personalized peptide vaccination:a new approach for advanced cancer as therapeutic cancer vaccine,Cancer Immunol.Iother,62(5):919-929
Siegel R.L.,Miller K.D.,and Jemal A.,2015,Cancer statistics2015,CA Cancer J.Clin.,65(1):5-29
Sun M.F.,Qin Z.H.,Xie M.Q.,Yuan J.F.,Lv M.N.,Yu J.S.,Wu C.Y.,and Cai J.P.,2010,Effect of different expression vectors temperatures on the activity of Eimeria tenella MalonylCo A:ACP transacylase,Xumu Shouyi Xuebao(Chinese Journal of Animal and Veterinary Sciences),41(9):1159-1165(孙铭飞,覃宗华,谢明权,袁建丰,吕敏娜,余劲术,吴彩艳,蔡建平,2010,不同表达条件对Eimeria tenella丙二酰单酰辅酶A:ACP转移酶活性的影响,畜牧兽医学报,41(9):1159-1165)
Su P.,and Gong G.L.,2017,Research progress on optimizing the expression of exogenous proteins in Escherichia coli,Sheng wu Jishu Tongbao(Biotechnology Bulletin),33(2):16-23(苏鹏,龚国利,2017,优化大肠杆菌表达目的蛋白的研究进展,生物技术通报,33(2):16-23)
Wei J.,Yi Z.,Wei Y.R.,Wang H.F.,Hu E.M.X.,Fu Z.H.,and Ran D.L.,2009,Effect of induced concentration and time of IPTG on the expression of VP1 protein of FMDV type AsiaⅠ,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),36(5):63-65(魏婕,易忠,魏玉荣,王海烽,胡尔玛西,符子华,冉多良,2009,IPTG诱导浓度、时间对AsiaⅠ口蹄疫病毒VP1蛋白表达的影响,中国畜牧兽医,36(5):63-65)
Yang S.J.,2000,Cholera toxin,Shengwu Jishu Tongxun(Letter in Biotechnology),11(4):292-295(杨淑静,2000,霍乱毒素,生物技术通讯,11(4):292-295)
Ye W.,2001,Cholera toxin can be selected to enhance the antigen presenting of dendritic cells to intranasally immunized CTL epitopes,Guowai Yixue(Foreign Medical Sciences),24(3):42-43(叶巍,2001,霍乱毒素可选择性增强树突细胞对鼻内免疫CTL表位的抗原呈递,国外医学(微生物学分册),24(3):42-43)
Zheng J.,Shen H.,and Chen Q.,2008,Optimizing expression of recombination metalloprotease of vibrio vulnificus in E.coli BL21/DE3,Shuli Yiyaoxue Zazhi(Journal of Mathematical Medicine),21(3):276-279(郑晶,申洪,陈清,2008,基于正交实验优化创伤弧菌金属蛋白酶融合蛋白的表达,数理医药学杂志,21(3):276-279)
Dvorak P.,Chrast L.,Nikel P.I.,Fedr R.,Soucek K.,Sedlackova M.,Chaloupkova R.,Lorenzo V.,Prokop Z.,and Damborsky J.,2015,Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3)carrying a synthetic metabolic pathway,Microbial Cell Factories.,14(1):201-215
Halin S.,Rudolfsson S.H.,Rooijen N.V.,and Bergh A.,2009,Extratumoral macrophages promote tumor and vascular growth in an orthotopic rat prostate tumor model,Neoplasia.,11(2):177-186
Jin Y.C.,Liu L.,Su X.Y.,Li J.P.,Li H.D.,and Zhou J.H.,2006,Effect of induce concentration,time and temperature of IPTG on the expression of the GST-GnRH/TRS gene,Heilongjiang Xumu Shouyi(Heilongjiang Animal Science and Veterinary Medicine),(8):17-19(金元昌,刘利,苏晓艳,李景鹏,李会东,周建红,2006,IPTG诱导浓度、时间及温度对重组促性腺激素释放激素基因表达的影响,黑龙江畜牧兽医,(8):17-19)
Kawamura Y.I.,Kawashima R.,Shirai Y.,Kato R.,Hamabata T.,Yamamoto M.,Fuukawa K.,Fujihashi K.,Mcghee J.R.,Hayashi H.,and Dohi T.,2003,Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB Translation,Eur.J.Immunol.,33(11):3205-3212
Li W.B.,2013,The research of expression of YAP in prostate cancer and the role of interaction between YAP and AR in CRPC transforming,Thesis for M.S.,Chongqing Medical University,Supervisor:Wang D.L.,pp.12-15(李文宾,2013,Yes相关蛋白在前列腺中的表达及其与AR相互作用在CRPC转化中机制的初步探讨,硕士学位论文,重庆医科大学,导师:王德林,pp.12-15)
Noguchi M.,Sasada T.,and Itoh K.,2013,Personalized peptide vaccination:a new approach for advanced cancer as therapeutic cancer vaccine,Cancer Immunol.Iother,62(5):919-929
Siegel R.L.,Miller K.D.,and Jemal A.,2015,Cancer statistics2015,CA Cancer J.Clin.,65(1):5-29
Sun M.F.,Qin Z.H.,Xie M.Q.,Yuan J.F.,Lv M.N.,Yu J.S.,Wu C.Y.,and Cai J.P.,2010,Effect of different expression vectors temperatures on the activity of Eimeria tenella MalonylCo A:ACP transacylase,Xumu Shouyi Xuebao(Chinese Journal of Animal and Veterinary Sciences),41(9):1159-1165(孙铭飞,覃宗华,谢明权,袁建丰,吕敏娜,余劲术,吴彩艳,蔡建平,2010,不同表达条件对Eimeria tenella丙二酰单酰辅酶A:ACP转移酶活性的影响,畜牧兽医学报,41(9):1159-1165)
Su P.,and Gong G.L.,2017,Research progress on optimizing the expression of exogenous proteins in Escherichia coli,Sheng wu Jishu Tongbao(Biotechnology Bulletin),33(2):16-23(苏鹏,龚国利,2017,优化大肠杆菌表达目的蛋白的研究进展,生物技术通报,33(2):16-23)
Wei J.,Yi Z.,Wei Y.R.,Wang H.F.,Hu E.M.X.,Fu Z.H.,and Ran D.L.,2009,Effect of induced concentration and time of IPTG on the expression of VP1 protein of FMDV type AsiaⅠ,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),36(5):63-65(魏婕,易忠,魏玉荣,王海烽,胡尔玛西,符子华,冉多良,2009,IPTG诱导浓度、时间对AsiaⅠ口蹄疫病毒VP1蛋白表达的影响,中国畜牧兽医,36(5):63-65)
Yang S.J.,2000,Cholera toxin,Shengwu Jishu Tongxun(Letter in Biotechnology),11(4):292-295(杨淑静,2000,霍乱毒素,生物技术通讯,11(4):292-295)
Ye W.,2001,Cholera toxin can be selected to enhance the antigen presenting of dendritic cells to intranasally immunized CTL epitopes,Guowai Yixue(Foreign Medical Sciences),24(3):42-43(叶巍,2001,霍乱毒素可选择性增强树突细胞对鼻内免疫CTL表位的抗原呈递,国外医学(微生物学分册),24(3):42-43)
Zheng J.,Shen H.,and Chen Q.,2008,Optimizing expression of recombination metalloprotease of vibrio vulnificus in E.coli BL21/DE3,Shuli Yiyaoxue Zazhi(Journal of Mathematical Medicine),21(3):276-279(郑晶,申洪,陈清,2008,基于正交实验优化创伤弧菌金属蛋白酶融合蛋白的表达,数理医药学杂志,21(3):276-279)
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