AIM:To obtain the entire gene open reading frame(ORF)and to construct the expression vectors for recombinantallergen production.METHODS:Gene fragments corresponding to the genespecific region and the cDNA ends of pollen allergens ofshort ragweed(Rg,Ambrosia artemisiifolia L.)were obtainedby pan-degenerate primer-based PCR and rapid amplificationof the cDNA ends(RACE),and the products were mixed toserve as the bridging PCR(BPCR)template.The full-lengthgene was then obtained.Partially overlapping primer-basedPCR(POP-PCR)method was developed to overcome theother problem,i.e.,the non-specific amplification of the ORFwith routine long primers for expression insert decoration.Northern blot was conducted to confirm pollen sources ofthe gene.The full-length coding region was evaluated forits gene function by homologue search in GenBank databaseand Western blotting of the recombinant protein Amb a 8(D106)expressed in Escherichia coli pET-44 system.RESULTS:The full-length cDNA sequence of Arab a 8(D106)was obtained by using the above procedure and deducedto encode a 131 amino acid polypeptide.Multiple sequencealignment exhibited the gene D106 sharing a homology ashigh as 54-89% and 79-89% to profilin from pollen andfood sources,respectively.The expression vector of the allergengene D106was successfully constructed by employing thecombined method of BPCR and POP-PCR.Recombinantallergen rAmb a 8(D106)was then successfully generated.The allergenicity was hallmarked by immunoblotting withthe allergic serum samples and its RNA source was confirmedby Northern blot.CONCLUSION:The combined procedure of POP-PCR andBPCR is a powerful method for full-length allergen generetrieval and expression insert decoration,which would beuseful for recombinant allergen production and subsequentdiagnosis and immunotherapy of pollen and food allergy. AIM: 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 genes 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 toserve as the bridging PCR (BPCR) template. The full-lengthgene was then obtained. Partially overlapping primer-based PCR ) was developed to overcome the other problem, ie, 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 genes 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 Arab a 8 (D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment showed the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. expression vector of the allergengene D106was successfully constructed by employing thecombined method of BPCR and POP-PCR. Recombinantallergen 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. CONCLUSION: The combined procedure of POP-PCR and PCR is a powerful method for full-length allergen generetrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy.