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Expression of bovine interleukin 15 and evaluation of its biological activity in vitro
N Vijay1, J Lijo1, H J Dechamma1
1FMD Research Laboratory, Indian Veterinary Research Institute, Hebbal, Bangalore, Karnataka, India.
Insights
Bovine interleukin-15 (IL-15) was cloned and expressed, showing biological activity. This cytokine enhances memory T cell survival, suggesting its use as a vaccine adjuvant to boost immunity.
Area of Science:
- Veterinary Immunology
- Molecular Biology
- Vaccinology
Background:
- Interleukin-15 (IL-15) is crucial for CD8(+) T cell development and memory formation.
- Understanding bovine IL-15 (bIL-15) function is key for improving vaccine efficacy in cattle.
Purpose of the Study:
- To clone and express functional bovine IL-15 (bIL-15).
- To investigate the role of bIL-15 in activating bovine CD8(+) T cells.
- To evaluate bIL-15 as a potential adjuvant for enhancing vaccine-induced immunity.
Main Methods:
- Amplification and cloning of bIL-15 coding and mature sequences.
- Expression and purification of bIL-15 fusion protein.
- Analysis of protein expression via SDS-PAGE and Western blotting.
- Assessment of biological activity using qPCR to measure Bcl2, STAT3, STAT5a, and CPT1a levels in stimulated PBMCs.
Main Results:
- Successfully amplified and cloned bIL-15 sequences.
- Purified bIL-15 fusion protein (32kDa) demonstrated biological activity.
- bIL-15 significantly upregulated Bcl2 (5-fold), STAT3 (9-fold), and STAT5a (10-fold) mRNA levels.
- bIL-15 increased Carnitine Palmitoyl Transferase 1a (CPT1a) expression by 2.5-fold, indicating enhanced memory T cell survival.
Conclusions:
- Bovine IL-15 was successfully cloned, expressed, and confirmed to be biologically active.
- The observed increase in CPT1a suggests bIL-15 promotes memory T cell survival.
- bIL-15 holds potential as an adjuvant in viral vaccines to prolong immunity in cattle.
Background/Aim:
Recent studies have shown that interleukin-15 (IL-15)is a critical factor for the development and proliferation of CD8(+) memory T cells. The aim of present study is to study the role bovine IL-15 (bIL-15)in activation pathway of bovine CD8(+) T cells if any, which will be useful in designing the adjuvant to increase the duration of immunity of the vaccine preparations.
Materials And Methods:
Coding region of bIL-15 (489) was amplified from cDNA of lipopolysaccharide-induced bovine peripheral blood mononuclear cells (PBMCs) using gene specific primers and cloned into pcDNA3.1(+). Mature length of bIL-15 was amplified using gene specific primers and cloned into pET32a for expression studies. Expressed fusion protein was purified using Ni-Nitrilotriacetic acid agarose affinity chromatography and analyzed by SDS-Polyacryamide gel electrophoresis (PAGE) and western blotting. Biological activity of purified protein was analyzed by quantitative Polymerase Chain Reaction (qPCR) for an increase in levels of Bcl2, STAT3 and STAT5a using cDNA synthesized from RNA of PBMCs induced with different concentrations of purified bIL-15. Role of IL-15 in inducing memory CD8(+) T cells was analyzed by qPCR for increase in the level of Carnitine Palmitoyl Transferase 1a (CPT1a) using cDNA synthesized from RNA of PBMCs induced with different concentrations of purified bIL-15.
Results:
Bovine IL-15 was amplified and analyzed by agarose gel electrophoresis, which showed a specific product of ~490bp, mature sequence was amplified using full-length as a template to get a product of ~350bp. The protein was expressed, purified and analyzed by SDS-PAGE and Western blotting, which showed a specific product of 32kDa. Biological activity of purified bIL-15 fusion protein showed an increase in levels of Bcl2, STAT3 and STAT5a with 5 fold, 9 fold, and 10 fold increases as analyzed by qPCR, respectively. Role of IL-15 in inducing memory T cells showed an increase in expression level of CPT1a at 2.5 fold increase as compared to control cells.
Conclusion:
Bovine IL-15 has been successfully cloned and expressed in our work, and the biological activity shows that the purified fusion protein is biologically active. As there is an increase in levels of CPT1a an enzyme critical for survival of memory T cells, IL-15 can be used for increase in the memory response, which can be used as an adjuvant with viral vaccines for increasing the immunity.
