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

Abstract

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 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.

Conclusion: 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.

Figure 1

Experimental design and steps involved in bridging PCR (BPCR) and partially overlapping primer-based PCR (POP-PCR). Gene fragments A and B had the junction region C. Under specific temperature conditions, the fragments would anneal together to form the template molecule, thus triggering the amplification cascade. The partially overlapping primers at each site of the template ends contained two primers pre-mixed in a certain molar ratio that had sequence identity but with different lengths. All the short primers located at the out-ward overhang region. The sequences of all primers used are shown in Table 1.

Figure 2

cDNA sequence of novel allergen gene D106 from short ragweed pollens. The five shadowed regions represent the primer sites, Sg1p5, S5D106, S3D10, DEN3 and RAC3.

Figure 3

Phylogenetic relationships between newly obtained pollen allergen D106 (Amb a 8(D106)) and its cognates from GenBank as inferred from CLUSTAL W (1.82) alignment of the amino acid sequences. Amb a 8(D106) shared a homology as highly as 54%-89% with different pollen allergens (from mugwort CAD12862 to apple Q9XF41) and as 79%-89% with different food allergens (from peanut Q9SQI9 to peach Q8GT39).

Figure 4

Northern blot profile confirming RNA derivation of cDNA of the newly obtained gene D106. Lane 1, total RNA from calyx and pedicel; Lane 2, total RNA from pollen. Both lanes exhibit obvious bands, suggesting the real RNA derivation of D106.

Figure 5

Agarose gel electrophoresis of PCR products. The PCR steps were depicted in Figure 1. Template in lanes 1, 2 and 5 is the mixture from gene fragments A and B; while tem-plate in lanes 3 and 4 is the BPCR product (lane 5). Lane 1, primer Sg1P5/RAC3; lane 2, primer POP51/POP31; lane 3, POP-PCR with primer POP51+POP52/POP31+POP32; lane 4, general PCR of the ORF of D106 with primer pair Sg1P5/DEN3; lane 5, BPCR of full-length D106 with primer Sg1P5/RAC3; M, molecular marker.

Figure 6

SDS-PAGE analysis (150 g/L) for expression (panel A) and immunoblot of fusion pollen allergen D106 (panel B). Panel A: 1. Lysate pellet of induced cells; 2. Cells without IPTG induction; 3. Supernatant of induced cell lysate; 4. Purified fusion pollen allergen D106; M. Broad range prestained SDS-PAGE standards (Bio-Rad, Hercules, CA, USA). Panel B: Fusion protein D106 was blotted with 8 serum samples from pollen allergic patients. Each strip represents one serum sample.