摘要
Objective: To study the function of the first extracellular membrane loop of the NH2-terminal of chemokine receptor 5(CCRS), and produce its specific antibody F(ab’ )2. Methods: Some amino acid sequences of β chemokine superfamily receptors were aligned by computer and the least homologous domain of the extracellular loops was located. In this paper, the first extracellular domain (114 nucleotides) was defined and amplified by PCR, and a expression vector of the recombinant GST fusion protein,pGEXIN/NR5, was constructed. After the inserted sequence was confirmed correct, the transformation and expression of fusion protein was performed in E. coli and the fusion protein was purified. Then 2 Newzealand rabbits were immunized. The anti-CCR5 NH2-terminal antibody F(ab’ )2 was made by protein A affinity chromatography, pepsin digestion and sepharose-12 column chromatography. Results: Reduced and unreduced SDS-PAGE and FAX analysis demonstrated that this F(ab’ )2 had a high specificity to combine with CCR5. Conclusion: A simple and quick method to prepare specific antibody F(ab’ )2 of certain functional domain is showed in this paper, by which we can get an improtant experiment material for studying gene expression, and it will provide a good idea and a technique to study other high similar superfamily members.
Objective: To study the function of the first extracellular membrane loop of the NH2-terminal of chemokine receptor 5(CCRS), and produce its specific antibody F(ab' )2. Methods: Some amino acid sequences of β chemokine superfamily receptors were aligned by computer and the least homologous domain of the extracellular loops was located. In this paper, the first extracellular domain (114 nucleotides) was defined and amplified by PCR, and a expression vector of the recombinant GST fusion protein,pGEXIN/NR5, was constructed. After the inserted sequence was confirmed correct, the transformation and expression of fusion protein was performed in E. coli and the fusion protein was purified. Then 2 Newzealand rabbits were immunized. The anti-CCR5 NH2-terminal antibody F(ab' )2 was made by protein A affinity chromatography, pepsin digestion and sepharose-12 column chromatography. Results: Reduced and unreduced SDS-PAGE and FAX analysis demonstrated that this F(ab' )2 had a high specificity to combine with CCR5. Conclusion: A simple and quick method to prepare specific antibody F(ab' )2 of certain functional domain is showed in this paper, by which we can get an improtant experiment material for studying gene expression, and it will provide a good idea and a technique to study other high similar superfamily members.
