Prokaryotic Expression, Purification, and Functional Characterization of the Large Yellow Croaker (Larimichthys crocea) Mannose Receptors Subunits (MRC1 and MRC2)
Prokaryotic Expression, Purification, and Functional Characterization of the Large Yellow Croaker (Larimichthys crocea) Mannose Receptors Subunits (MRC1 and MRC2)
Xiangli Dong1,2, Shilin Mikhail Borisovich2, Jiji Li1,*, Jianyu He1, Zeqin Fu1, Yingying Ye1, Julia N. Lukina3, Olga V. Apalikova4 and Jianshe Zhang1
ABSTRACT
The mannose receptor (MR) is an important receptor for the innate immune response. It is a member of the C-type lectin domain family, and has two subunits, MRC1 and MRC2. The MRC1 and MRC2 cDNA sequences have been analyzed and characterized in the large yellow croaker, a highly valued, farm raised fish that is vulnerable to many infections. Quantitative real-time PCR (qRT-PCR) analysis indicated that MRC1 and MRC2 mRNAs were expressed in eight different large yellow croaker tissues, and that their expression was up-regulated by Vibrio anguillarum challenge. Here, we performed membrane protein analysis and epitope analysis to select MRC1 and MRC2 protein fragments suitable for antibody production. We then PCR amplified L.c-MRC1 and L.c-MRC2 and cloned them into prokaryotic protein expression vectors (MRC1 (1044bp)-pET32A and MRC2 (993bp)-pET32A). We performed SDS-PAGE analysis of the expressed L.c-MRC1 and L.c-MRC2 proteins and demonstrated high protein expression levels and purity. This study generates some essential molecular biology tools for the study of L. crocea MRC1 and MRC2 protein structure and function. These tools will enable us to better understand the biological functions of MRC1 and MRC2 in defending against pathogenic bacteria challenge and the innate immune response in the large yellow croaker. These findings also provide a foundation for the preparation of a Vibrio vaccine.
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