Basic Research
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2004; 10(14): 2103-2108
Published online Jul 15, 2004. doi: 10.3748/wjg.v10.i14.2103
Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration
Ai-Lin Tao, Shao-Heng He
Ai-Lin Tao, Shao-Heng He, Allergy and Inflammation Research Institute, Medical College, Shantou University, Shantou 515031, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Li Ka Shing Foundation, Hong Kong, China, No. C0200001 and Natural Science Foundation of Guangdong Province, No. 034617
Correspondence to: Professor Shao-Heng He, Allergy and Inflammation Research Institute, Medical College, Shantou University, 22 Xin-Ling Road, Shantou 515031, Guangdong Province, China. shoahenghe@hotmail.com
Telephone: +86-754-8900405 Fax: +86-754-8900192
Received: January 15, 2004
Revised: February 10, 2004
Accepted: March 2, 2004
Published online: July 15, 2004
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.

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