Cloning and sequencing of the cDNA encoding a human differentiation antigen

Objective Cloning and sequencing of the cDNA encoding a differentiation antigen 5C5. Methods Construction of a λgt11 cDNA library of 3D5 cells, a human activated B cell line; isolation of positive cDNA clones from the cDNA library with phage hybridization; sequencing of the cDNA; open reading frame analysis; hydrophobic analysis of the amino acid sequence of the deduced protein; determination of the length of 5C5 mRNA transcript with Northern blot hybridization test; determination of 5C5 mRNA transcription in various cell lines with RT PCR. Results 5C5 1 cDNA (0.9kb) and 5C5 2 cDNA (0.7kb) have been isolated from a λgt11 cDNA library of 3D5 cell, a human activated B cell line, by using a 467bp 5C5 cDNA as a probe. Nucleotide sequence analysis has shown that the 5C5 1 cDNA is 905bp long with an open reading frame (ORF) from 19 to 555bp in it. The TopPred 2 topology prediction of the membrane protein suggests that the 179aa protein deduced from the ORF has a helix membrane spanning segment from 94 to 114aa. The orientation is that the N terminal is inside the cell. 5C5 2 cDNA is 754bp long with an ORF from 163 to 459bp encoding a protein with a length of 99aa. Northern blotting shows two mRNA transcripts with lengths of 0.9kb and 0.7kb respectively, indicating that the 905bp cDNA is a full length cDNA. RT PCR reveals strong, medium and weak expressions of 5C5 mRNA in 3D5 cell, Daudi cell and Jurkat cell respectively. Conclusion The 905bp 5C5 1 cDNA is a full length cDNA encoding a human membrane protein.