Molecular characterization, expression patterns, and immune functions of snakehead interleukin-21/receptor (shIL-21/shlL-21R)

Abstract

Interleukin-21 (IL-21), a γ-chain family cytokine, plays a crucial role in mammalian immune regulation, but its function in fish remains poorly understood. This study successfully cloned and identified the IL-21 gene (shIL-21) in Northern snakehead (*Channa argus*), systematically analyzed its molecular characteristics, tissue expression profile, and dynamic changes during bacterial infection, and validated the functional roles of recombinant shIL-21 protein in promoting leukocyte proliferation, activating the JAK-STAT pathway, and inducing key inflammatory factor expression. The findings lay an important foundation for developing detection kits to monitor IL-21/IL-21R signaling pathway activity in fish.

I. Introduction

IL-21 is primarily produced by activated CD4⁺ T cells and NKT cells. By binding to the IL-21R/γc receptor complex, it activates signaling pathways such as JAK-STAT, regulating the functions of various immune cells, including B cells, T cells, and dendritic cells. The Northern snakehead is an important freshwater economic fish in China, often threatened by infections from *Nocardia seriolae* and *Aeromonas schubertii*. Research on its IL-21/IL-21R system is of significant value for elucidating immune mechanisms and developing related detection tools.

II. Cloning and Bioinformatics Analysis of shIL-21 Gene

The shIL-21 open reading frame is 438 bp in length, encoding 145 amino acids with a predicted protein molecular weight of 16.50 kDa. The protein exhibits typical IL-21 family structural features, including four conserved cysteine residues and four α-helices (Figure 1). Phylogenetic analysis revealed that shIL-21 is most closely related to the IL-21 of *Lates calcarifer* (66.44% homology) and forms an independent evolutionary branch among teleost fish (Figure 2), suggesting high conservation of this gene during evolution.

III. Expression Characteristics of shIL-21 in Healthy and Infected States

Tissue expression profiling showed that shIL-21 is widely expressed in healthy Northern snakehead tissues, with the highest expression in the liver, followed by the gill, head kidney, and spleen (Figure 3). After infection with *Nocardia seriolae* and *Aeromonas schubertii*, shIL-21 expression was significantly upregulated in the head kidney and spleen, showing time-dependent changes (Figure 4). In vitro experiments further confirmed that pathogen-associated molecular patterns (PAMPs) such as lipoteichoic acid (LTA), lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (poly(I:C)) could induce upregulated shIL-21 expression in head kidney leukocytes (Figure 5).

IV. Recombinant shIL-21 Protein Expression and Functional Validation

Recombinant shIL-21 protein was successfully obtained using a prokaryotic expression system (Figure 6). Functional experiments demonstrated that this protein significantly promotes head kidney leukocyte proliferation, increasing the proliferation rate from 18.57% to 30.50% after 48 hours of stimulation (Figure 7). Additionally, rshIL-21 induced the upregulation of key genes in the JAK-STAT pathway (JAK1, JAK3, STAT1, STAT3), endogenous shIL-21, and pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β) (Figure 8), suggesting that shIL-21 participates in immune regulation by activating the JAK-STAT signaling pathway.

V. Discussion and Future Perspectives

This study is the first to identify an IL-21 homologous gene in Northern snakehead, confirming its important role in bacterial infection immunity and preliminarily elucidating its functional mechanism in regulating leukocyte proliferation and inflammatory responses via the JAK-STAT pathway. These results provide a theoretical basis and molecular targets for developing detection kits to assess IL-21/IL-21R signaling activity, evaluate immune status, and diagnose bacterial infections in fish. Future research could focus on cloning and interaction studies of the IL-21 receptor (shIL-21R) to improve the detection system for this signaling pathway and advance the development and application of immune monitoring tools for aquatic animals.

VI. Which Manufacturers Provide IL21/IL21R Detection Kits?

Nanjing UA-Bio Technology Co., Ltd. has independently developed the "Human IL21/IL21R Binding Kit", a high-performance in vitro detection platform specifically designed for studying key pathways in cellular immune regulation and lymphocyte function. This kit is engineered to accurately and efficiently quantify the binding activity between interleukin-21 (IL-21) and its specific receptor IL-21R, providing a stable and reliable standardized solution for drug screening, mechanism research, and efficacy evaluation in fields such as tumor immunology, autoimmune diseases, and immune cell therapy.

Nanjing UA-Bio Technology Co., Ltd. is dedicated to providing cutting-edge, high-quality core reagents and tools for immunology, cell therapy, and innovative drug development. For detailed technical specifications, validation data, or specific application inquiries regarding the "Human IL21/IL21R Binding Kit" (Catalog No.: UA086015), please feel free to contact us.