| Autor: |
Lalor, P., Bucci, C., Fornaro, M., Rattazzi, M.C., Nakauchi, H., Herzenberg, L.A., Alberti, S. |
| Předmět: |
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| Zdroj: |
Immunology; May92, Vol. 76 Issue 1, p95-102, 8p |
| Abstrakt: |
We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8α). A bovine thymus cDNA library was hybridized at Iow stringency with a human CD8α cDNA clone. The first round of screening of 5 × 104 independent colonies yielded 12 clones containing incomplete BoCD8α, genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8α clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8α clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8α. Sequence analysis confirmed that the clone under study encoded the BoCD8α. The overall similarity of the BoCD8α coding region with the human CD8α coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8α. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among ali species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8α to the α1 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8α clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8α gene, we constructed a chimeric gene containing the coding sequence of the BoCD8α clone and synthetic sequences corresponding to the first two amino acids of the human CD8α leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8α construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8α. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8α gene, as previously suggested on serological grounds. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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