Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration.

OBJECTIVE

To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production.

METHODS

Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-length coding region was evaluated for its gene function by homologue search in GenBank database and Western blotting of the recombinant protein Amb a 8(D106) expressed in Escherichia coli pET-44 system.

RESULTS

The full-length cDNA sequence of Amb a 8(D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment exhibited the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. Recombinant allergen rAmb a 8(D106) was then successfully generated. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot.

CONCLUSIONS

The combined procedure of POP-PCR and BPCR is a powerful method for full-length allergen gene retrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy.