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Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle
Published on: July 11, 2015
[Expression and antiviral assay of bovine interferon-gamma]
Zhengzhong 'u1, Xiang Chen, Fengli Shan
1Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China.
Insights
Researchers successfully expressed bovine interferon-gamma (BoIFN-gamma) using various expression systems, including E. coli and insect cells. The study also developed a sandwich ELISA for quantifying BoIFN-gamma, aiding clinical applications.
Area of Science:
- Molecular Biology
- Immunology
- Biotechnology
Background:
- Bovine interferon-gamma (BoIFN-gamma) plays a crucial role in the bovine immune response.
- Efficient expression and characterization of recombinant BoIFN-gamma are essential for its therapeutic and diagnostic applications.
Purpose of the Study:
- To clone and express the BoIFN-gamma gene in multiple expression systems.
- To characterize the expressed recombinant BoIFN-gamma.
- To establish a reliable method for quantifying BoIFN-gamma.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) for gene amplification.
- Cloning into bacterial (pET-30a+, pGEX-6p-1) and baculovirus vectors.
- Transfection into COS-7 and Sf9 insect cells.
- SDS-PAGE, indirect immunofluorescence assay, and antiviral activity assays.
- Sandwich ELISA development using monoclonal antibodies.
Main Results:
- Recombinant BoIFN-gamma was successfully expressed in COS-7 cells, E. coli (soluble forms at 23 kDa and 43 kDa), and Sf9 insect cells.
- Antiviral activities of recombinant proteins were quantified.
- A sandwich ELISA was established for sensitive detection and quantification of BoIFN-gamma.
Conclusions:
- The study demonstrates successful expression of functional BoIFN-gamma in diverse systems.
- The developed sandwich ELISA provides a valuable tool for BoIFN-gamma research and clinical diagnostics.
Abstract:
Bovine interferon-gamma (BoIFN-gamma) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with ConA. The products of RT-PCR were cloned into pVAX1 vector, positive recombinant clone was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pVAX1-BolFN-gamma was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-gamma was expressed in COS-7 cells. BoIFN-gamma gene (without signal peptide) was cloned into pET-30a(+) and pGEX-6p-1 vector, and transformed into the Escherichia coli cells. After optimizing the induction condition, SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively. BoLFN-gamma precursor gene (with signal peptide) was cloned into transfer vector pFastBac 1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-gamma gene was integrated into shuttle vector Bacmid, and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus. Indirect immunofluorescent assay analysis confirmed that rBac-BoLFN-gamma was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-gamma, rGST-BoIFN-gamma and rBac-BoIFN-gamma were up to 8.389 x10(7) U/mg, 6.554 x10(5) U/mg and 4.096 x 10(4) U/mL respectively, which were analyzed in MDBK/VSV system. A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4, which can detect BoIFN-gamma in quantity and provide a useful method for the clinical practice and research of BolFN-gamma.
