Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequ...Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So we explored a condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody.Methods: Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta(DE3) E.coli. Different concentrations of IPTG(0.05 and 0.5 mmol/L) and different culture temperatures(16 and37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution(15, 60, and300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbits. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western blot assays.Results: Under the conditions, IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta(DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbits. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines.Conclusion: An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blotting.展开更多
基金supported by the CAMS Innovation Fund for Medical Sciences(CIFMS)(2016-I2M-2-003)National Standardization Project of Traditional Chinese Medicine(ZYBZH-Y-JIN-34)
文摘Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So we explored a condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody.Methods: Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta(DE3) E.coli. Different concentrations of IPTG(0.05 and 0.5 mmol/L) and different culture temperatures(16 and37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution(15, 60, and300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbits. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western blot assays.Results: Under the conditions, IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta(DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbits. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines.Conclusion: An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blotting.
