Journal of Food Science and Biotechnology

Prokaryotic Expression , Purification , and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii Channelrhodopsin 1

Jiurong WANG, State Key Laboratory of Food Nutrition and Safety , Tianjin University of Science & Technology , Tianjin 300457 , China
Bin XUE, College of Biotechnology and Bioengineering , Zhejiang University of Technology , Hangzhou 310014 , China
Zhenchuan FAN, State Key Laboratory of Food Nutrition and Safety , Tianjin University of Science & Technology , Tianjin 300457 , China;National International Science and Technology Cooperation Institute of Health Biotechnology , Tianjin University of Science & Technology , Tianjin 300457 , China Follow

Corresponding Author(s)

樊振川（1974—），男，博士，教授，博士研究生导师，主要从事纤毛信号转导及营养代谢型纤毛病致病机理研究。E-mail：fanzhen@tust.edu.cn

Abstract

[Objective ] This study aims to prepare a highly specific antibody against channelrhodopsin- 1 (ChR 1) 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∷ChR 1 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 ChR 1 antiserum.ChR 1-specific IgG antibodies were enriched from the antiserum through protein A purification.The antibody specificity was assessed by immunoblotting,and the subcellular localization of ChR 1 was analyzed by immunofluorescence.[Result ] The results of indirect ELISA indicated that the ChR 1 antiserum had a titer of 1∶102 400.Immunoblotting with the purified ChR 1 antibody identified a distinct band corresponding to endogenous ChR 1 in CC- 125 cells,with no nonspecific band observed,thereby confirming the high specificity of the antibody.Immunofluorescence staining revealed clear localization of ChR 1 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 ChR 1 were achieved.This study lays a crucial foundation for delving into the localization of ChR 1 in C.reinhardtii and the molecular regulatory mechanisms of ChR 1 in phototaxis and negative phototaxis.

Publication Date

6-15-2025

First Page

Last Page

DOI

10.12441/spyswjs.20231010004

Recommended Citation

WANG, Jiurong; XUE, Bin; and FAN, Zhenchuan (2025) "Prokaryotic Expression , Purification , and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii Channelrhodopsin 1," Journal of Food Science and Biotechnology: Vol. 44: Iss. 6, Article 7.
DOI: 10.12441/spyswjs.20231010004
Available at: https://spsw.spyswjs.cnjournals.com/journal/vol44/iss6/7

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