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Expression and antibody generation of the cancer-testis antigen, BIOT2-S
1Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
Abstract:
Biot2-S is a mouse cancer-testis antigen gene that was identified using the cross-reactive serological analysis of recombinant cDNA expression libraries (SEREX) technique in the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. To express BIOT2-S and generate its antibody for further investigation, the Biot2-S prokaryotic recombinant expression vector Biot2-S/pGEX6P-1 was constructed with Escherichia coli DH5α as a cloning vector, and BIOT2-S was expressed in E. coli Rosetta (DE3). The recombinant BIOT2-S was expressed in the form of an inclusion body and the targeted recombinant BIOT2-S was produced at the level of approximately 25% total bacterial proteins after being induced with optimum conditions (0.2 mM isopropyl-β-D-thiogalactopyranoside for 6 h at 37°C). The target protein was purified by glutathione S-transferase (GST)-trap FF affinity chromatography and detected by western blot. The purified recombinant protein was further confirmed by electrospray ionization quadrupole time-of-flight mass spectrometry after removal of the GST-tags. Then the purified BIOT2-S was used to immunize adult rabbits to generate its antibody. The antibody was purified and its specificity determined. The titer of the antibody was shown to reach 10(4) and the antibody was demonstrated to be able recognize the corresponding protein in the testes of mouse and chicken; the tumor cell lines CT-26 and S180 also reacted with the antibody. This study provides a valuable foundation for further research on the cancer-testis antigen BIOT2-S.
Insights
Researchers successfully expressed and purified the Biot2-S cancer-testis antigen in bacteria. The generated antibody specifically recognized Biot2-S in mouse and chicken testes and in tumor cell lines, paving the way for further cancer research.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Cancer-testis antigens (CTAs) are promising targets for cancer immunotherapy.
- Biot2-S is a novel mouse CTA identified via SEREX.
- Further characterization and antibody generation are crucial for studying Biot2-S.
Purpose of the Study:
- To express the Biot2-S gene and produce the corresponding recombinant protein.
- To generate a specific antibody against Biot2-S.
- To validate the antibody's specificity and utility in detecting Biot2-S.
Main Methods:
- Construction of a prokaryotic recombinant expression vector (Biot2-S/pGEX6P-1) using E. coli.
- Expression and purification of recombinant Biot2-S using affinity chromatography.
- Antibody generation in rabbits and validation by Western blot and mass spectrometry.
Main Results:
- Recombinant Biot2-S was successfully expressed in E. coli Rosetta (DE3) as an inclusion body.
- Purified recombinant Biot2-S was confirmed by mass spectrometry.
- A specific antibody against Biot2-S was generated with a titer of 10^4, recognizing the antigen in mouse and chicken testes, and in CT-26 and S180 tumor cell lines.
Conclusions:
- The study successfully established a method for producing and purifying recombinant Biot2-S.
- A specific and functional antibody against Biot2-S was developed.
- This work provides a foundation for investigating the role of Biot2-S in cancer biology and its potential as a therapeutic target.
