摘要
Objective Using homology primers for clone of β-chemokine receptor supergene-family from lower level RNA expression. Methods: Through similarity alignment of the conservative transmembrane domainof human MCP-l receptor, mouse MIP-lα receptor and rat IL-8 receptor. the degenerate oligonucleotide homology primers were designed and a cDNA fragment was amplified by RT--PCR. After a new gene sequenceof chemokine receptor supergene-family in GenBank was identified, the new gene full sequence was cloned bya Marathon PCR of the specific primers. Results: Mouse CCR5 full-length cDNA with 2888 bp was cloned,including open reading frame 1065 oligonucleotides and coding 355 amino acids. This gene was a novel member of the β-chemokine receptor supergene-family confirmed by sequence analysis, ligand binding assay andinforming from GenBank. Conclusion: Using homology primers, the novel member of the supergene-familycan be captured. This method is a new cloning strategy and has extensive significance in the molecular cloningof a new gene.
Objective Using homology primers for clone of β-chemokine receptor supergene-family from lower level RNA expression. Methods: Through similarity alignment of the conservative transmembrane domainof human MCP-l receptor, mouse MIP-lα receptor and rat IL-8 receptor. the degenerate oligonucleotide homology primers were designed and a cDNA fragment was amplified by RT--PCR. After a new gene sequenceof chemokine receptor supergene-family in GenBank was identified, the new gene full sequence was cloned bya Marathon PCR of the specific primers. Results: Mouse CCR5 full-length cDNA with 2888 bp was cloned,including open reading frame 1065 oligonucleotides and coding 355 amino acids. This gene was a novel member of the β-chemokine receptor supergene-family confirmed by sequence analysis, ligand binding assay andinforming from GenBank. Conclusion: Using homology primers, the novel member of the supergene-familycan be captured. This method is a new cloning strategy and has extensive significance in the molecular cloningof a new gene.
