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Home > Archive>Volume , Issue 6, 2025 >59-65. DOI:10.12441/spyswjs.20231010004
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Prokaryotic Expression, Purification, and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii Channelrhodopsin 1
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1.State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China;2.College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China;3.National International Science and Technology Cooperation Institute of Health Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

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    Abstract:

    [Objective] This study aims to prepare a highly specific antibody against channelrhodopsin-1 (ChR1) from Chlamydomonas reinhardtii (C. reinhardtii) and investigate its regulatory mechanisms in cellular phototaxis and negative phototaxis.[Method] The recombinant expression vector pET-28a(+)-chr1 was constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells. The expression of the fusion protein 6×His∷ChR1 was induced with IPTG, and the obtained protein was purified by nickel-affinity chromatography. The purified protein was used to immunize New Zealand white rabbits for the production of ChR1 antiserum. ChR1-specific IgG antibodies were enriched from the antiserum through protein A purification. The antibody specificity was assessed by immunoblotting, and the subcellular localization of ChR1 was analyzed by immunofluorescence.[Result] The results of indirect ELISA indicated that the ChR1 antiserum had a titer of 1∶102 400. Immunoblotting with the purified ChR1 antibody identified a distinct band corresponding to endogenous ChR1 in CC-125 cells, with no nonspecific band observed, thereby confirming the high specificity of the antibody. Immunofluorescence staining revealed clear localization of ChR1 at the eyespot and specific staining, demonstrating the suitability of the antibody for immunofluorescence assays.[Conclusion] The prokaryotic expression, purification, and polyclonal antibody preparation of ChR1 were achieved. This study lays a crucial foundation for delving into the localization of ChR1 in C. reinhardtii and the molecular regulatory mechanisms of ChR1 in phototaxis and negative phototaxis.

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WANG Jiurong, XUE Bin, FAN Zhenchuan. Prokaryotic Expression, Purification, and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii Channelrhodopsin 1[J]. Journal of Food Science and Biotechnology,2025,(6):59-65.

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History
  • Received:October 10,2023
  • Revised:December 12,2023
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  • Online: October 11,2025
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