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Published on: November 3, 2014
Cloning, heterologous expression, and polyclonal antibody generation against the macrophage mannose receptor gene of
Jing Chen1, Lingzhi Tang2, Hongbo Li1
1Department of Biochemistry and Molecular Biology School of Medicine, Jishou University, Jishou, Hunan, 416000, China; Hunan Provincial Key Laboratory for Synthetic Biology of Traditional Chinese Medicine, Biomedical Research Institute, Hunan University of Medicine, Huaihua, Hunan, 418000, China.
Abstract:
The macrophage mannose receptor (MMR) is an important member of the C-type lectin family. It plays a significant role in recognizing pathogen-associated glycans and mediating immune phagocytosis. However, its specific function in insect immunity remains elusive. To investigate the expression and function of MMR genes in Lepidoptera, this study focused on the cabbage white butterfly, Pieris rapae. We cloned the P. rapae MMR gene and achieved its recombinant expression in Escherichia coli. Following purification and refolding, soluble recombinant MMR protein was obtained. An immunogenic peptide derived from the protein sequence was synthesized and used to immunize New Zealand White rabbits. High-titer, affinity-purified polyclonal antibody was subsequently isolated. The antibody demonstrated high specificity and sensitivity, making it suitable for the quantitative detection of endogenous MMR protein in P. rapae. This study established a strategy for producing soluble recombinant MMR protein and generated a highly specific polyclonal antibody, thereby providing a foundational platform for the detection of MMR proteins, functional studies, and the elucidation of immune recognition mechanisms.
Insights
Researchers developed a method to detect the macrophage mannose receptor (MMR) in insects. This breakthrough enables further study of insect immunity and pathogen recognition mechanisms.
Area of Science:
- Immunology
- Molecular Biology
- Entomology
Background:
- The macrophage mannose receptor (MMR), a C-type lectin, is crucial for pathogen recognition and phagocytosis in vertebrates.
- Its role in insect immunity, particularly in Lepidoptera, is not well understood.
Purpose of the Study:
- To investigate the expression and function of MMR genes in the cabbage white butterfly, Pieris rapae.
- To develop tools for detecting and studying MMR in insects.
Main Methods:
- Cloning and recombinant expression of the P. rapae MMR gene in E. coli.
- Purification and refolding of soluble recombinant MMR protein.
- Synthesis of an immunogenic peptide and generation of a high-titer polyclonal antibody in rabbits.
- Validation of antibody specificity and sensitivity for MMR detection.
Main Results:
- Successfully produced soluble recombinant P. rapae MMR protein.
- Generated a specific and sensitive polyclonal antibody against P. rapae MMR.
- Established a reliable method for quantitative detection of endogenous MMR protein in P. rapae.
Conclusions:
- This study provides a foundational platform for studying MMR function in insect immunity.
- The developed antibody and recombinant protein are essential tools for future research on immune recognition mechanisms in Lepidoptera.
- Elucidating MMR's role can advance our understanding of insect defense strategies.
