Recombinant Bubalus bubalis Somatotropin (GH1)
Description
Molecular Characterization and Production
Recombinant Bubalus bubalis Somatotropin (GH1) is produced via molecular cloning and expression systems. Key steps include:
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Gene Isolation: Partial sequences of the GH gene (including exons 3–4 and intronic regions) are amplified from buffalo liver RNA .
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Expression Systems: The gene is expressed in Escherichia coli BL21 (RIPL) Codon Plus, forming inclusion bodies. Refolding via cysteine-cystine redox potential restores biological activity .
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Structural Variations: Amino acid substitutions in the extracellular domain distinguish it from bovine GHR (e.g., specific residues in Nili-Ravi buffalo) .
Table 1: Key Molecular Features of Recombinant Bubalus bubalis GH1
Biological Activity and Mechanisms
Recombinant GH1 mediates effects through direct binding to growth hormone receptors (GHR) and indirect insulin-like growth factor-1 (IGF-1) upregulation:
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Receptor Binding: GH1 binds dimeric GHRs, activating JAK2-STAT pathways to regulate gene transcription .
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IGF-1 Synthesis: GH1 stimulates hepatic IGF-1 production, enhancing cellular proliferation and nutrient partitioning .
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Functional Validation: In vitro studies show recombinant buffalo GH-binding protein (GHBP) increases HeLa cell proliferation by 20–25% when co-administered with ovine GH .
Comparative Analysis with Bovine Somatotropin
While structural similarities exist between buffalo and bovine GH, functional differences arise from species-specific receptor interactions:
Table 2: Buffalo vs. Bovine Somatotropin
Applications in Buffalo Physiology
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Growth Enhancement: GH1 promotes linear bone growth and muscle mass via STAT5-mediated osteoblast differentiation .
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Lactation Efficiency: Increases mammary gland nutrient uptake, sustaining milk yield by 10–15% in treated buffaloes .
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Lipid Metabolism: Modulates fatty acid profiles in milk, reducing short-chain saturated fats while increasing long-chain unsaturated variants .
Research Gaps and Future Directions
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Long-Term Safety: No studies assess chronic exposure effects in humans or buffaloes.
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Species-Specificity: Molecular docking analyses suggest GH1 binds human IGF-1 receptors weakly, but cross-reactivity risks require validation .
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Environmental Impact: Gene flow studies in buffalo populations indicate potential for unintended genomic changes .
Product Specs
Target Background
Q&A
Experimental Design for Studying Recombinant Bubalus bubalis Somatotropin (GH1)
Q: How can researchers design experiments to study the effects of recombinant Bubalus bubalis somatotropin (GH1) on milk production in buffaloes?
A: To study the effects of recombinant Bubalus bubalis somatotropin (GH1) on milk production, researchers can employ a controlled trial design. This involves administering recombinant GH1 to a group of buffaloes and comparing their milk yield and composition with a control group. Key parameters to measure include milk volume, fat content, and protein levels. Additionally, gene expression analysis using techniques like differential display reverse transcriptase PCR (DDRT-PCR) can help identify genes involved in increased milk synthesis .
Data Analysis for Contradictory Results
Q: How can researchers analyze contradictory data when comparing the effects of recombinant Bubalus bubalis somatotropin (GH1) across different studies?
ELISA Methodology for GH1 Detection
Q: What are the key considerations for developing an ELISA to detect recombinant Bubalus bubalis somatotropin (GH1) in serum and milk samples?
A: Developing an ELISA for detecting recombinant Bubalus bubalis somatotropin (GH1) requires careful selection of antibodies, optimization of assay conditions, and validation through tests for sensitivity, specificity, and recovery. The use of affinity-purified antibodies raised against recombinant bovine somatotropin can enhance specificity. Additionally, ensuring parallelism between standard curves and sample dilutions is crucial for accurate quantification .
Gene Expression Profiling
Q: How can researchers use gene expression profiling to understand the molecular mechanisms underlying the effects of recombinant Bubalus bubalis somatotropin (GH1) on milk synthesis?
A: Gene expression profiling can be achieved through techniques like DDRT-PCR and quantitative real-time PCR. These methods allow researchers to identify differentially expressed genes following treatment with recombinant GH1. By analyzing the expression levels of candidate genes, researchers can elucidate the molecular pathways involved in enhanced milk production. This approach has revealed novel transcripts involved in milk synthesis, highlighting the complexity of the process .
Purification and Characterization of Recombinant GH1
Q: What methods are effective for purifying and characterizing recombinant Bubalus bubalis somatotropin (GH1) produced in E. coli?
A: Effective purification of recombinant GH1 involves a two-step process using hydrophobic interaction and ion-exchange chromatographies. This approach eliminates the need for ultrafiltration and dialysis, making it more efficient. Characterization can be performed using reverse-phase HPLC and non-reducing SDS-PAGE to distinguish between reduced and oxidized forms of the hormone. Biophysical and biochemical analyses ensure that the purified hormone is functionally identical to natural pituitary GH .
Screening for rbST Treatment
Q: How can researchers screen for recombinant bovine somatotropin (rbST) treatment in buffaloes using serum and milk samples?
A: Screening for rbST treatment can be effectively done using enzyme-linked immunosorbent assays (ELISAs) that measure serum responsiveness to rbST. An acid-stripping ELISA can be employed to analyze serum and milk samples. This method is suitable for compliance with regulatory standards and can be used independently without needing additional biomarkers .
Advanced Research Questions
Q: What are some advanced research questions that could be explored in the context of recombinant Bubalus bubalis somatotropin (GH1)?
A: Advanced research questions might include:
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Investigating the long-term effects of recombinant GH1 on buffalo health and milk quality.
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Exploring the genetic basis of variability in response to GH1 treatment among different buffalo breeds.
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Developing novel delivery systems to enhance the efficacy and stability of recombinant GH1.
These questions require sophisticated experimental designs and analytical techniques to address the complex interactions between GH1, genetics, and environmental factors.
Basic vs. Advanced Questions
Q: How can researchers distinguish between basic and advanced research questions related to recombinant Bubalus bubalis somatotropin (GH1)?
A: Basic questions typically focus on the fundamental aspects of recombinant GH1, such as its structure, function, and basic effects on milk production. Advanced questions delve into more complex issues, including molecular mechanisms, genetic variability, and long-term impacts on animal health. Advanced research often involves integrating multiple disciplines and employing cutting-edge methodologies to address these complex questions.
Example Data Table: Intra and Inter-Assay Variations for GH1 Measurement
| Sample Type | Mean GH1 (ng/ml) | Within Assay CV% | Between Assay CV% |
|---|---|---|---|
| Serum | 8.40 ± 0.74 | 3.36 | 8.81 |
| Serum | 12.00 ± 0.55 | 3.92 | 4.59 |
| Milk | 1.20 ± 0.17 | 6.74 | 12.31 |
