Expression and antibody generation of the cancer-testis antigen, BIOT2-S.
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.
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
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JY
Wang
Key Laboratory of the Sichuan Academy of Medical Sciences, Sichuan Provincial People's Hospital, Chengdu, China.
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M
Cao
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
M-R
MR
Guo
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
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S
Li
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
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XF
Yang
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China.
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M
Wang
Key Laboratory of the Sichuan Academy of Medical Sciences, Sichuan Provincial People's Hospital, Chengdu, China.
J
J
Fang
Key Laboratory of Biological Resources and Ecological Environments of The Chinese Education Ministry, College of Life Science, Sichuan University, Chengdu, China zj804@163.com.
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Zhao
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002645:Chickens; D002846:Chromatography, Affinity; D004926:Escherichia coli; D007106:Immune Sera / Q000096:biosynthesis / Q000737:chemistry*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D013737:Testis / Q000378:metabolism
D000951:Antigens, Neoplasm; C543854:Ccdc7b protein, mouse; D007106:Immune Sera; D011994:Recombinant Proteins