It would be more disturbing when the GC content of the open reading frame (ORF) ends greatly deviates from 50%, which would usually occur in the 5’ end of the ORF. The melting temperature (Tm) of such primers always goes beyond the ordinary limit of 72 ☌, and the amplification specificity hence is reduced. The other is the gene specific region for stringent polymerase chain reaction (PCR). One is the region for restriction recognition or the vector specific sequences for ligation-independent cloning (LIC), like newly developed pET-44 EK/LIC system. As for the expression vector construction, specific primers needed usually comprise two parts. Screening and detection of different clones obtained would account for the main expenditure of the prophase work of gene cloning. That is to say, though the length of amplified sequences is consistent with anticipation, the nucleotide compositions in different sequences are dramatically different and the existence of polymorphism is usually observed. In the process, a frequently occurred problem is the existence of alternatives in the amplification template.
Generally, many full-length genes are acquired by prior acquisition of the specific fragments, and subsequently constructed in conception with computers by joining the amphi-end sequences obtained by rapid amplification of cDNA end (RACE). Therefore, it is pivotal to obtain full-length allergen gene and to construct the expression vector. Allergen-specific immunotherapy (SIT) represents one of the few curative approaches towards allergy, for which a powerful method is to express the recombinant allergen proteins in vitro. Short ragweed (Rg, Ambrosia artemisiifolia L.) is an important source of airborne allergens all over the world and has become an immunodominant allergen in China. Patients with pollinosis have a high risk of developing a related food allergy, up to 70% of the patients are also allergic to fruits, vegetables or nuts. Among the “cross-reactivity” food allergies, the so-called pollen-related food allergies are the most important. The limitations for developing effective treatment regimens are due to some still unresolved and ambiguous aspects of the cross-reactivity of food and pollen allergy. In the past years, we have improved our ability to recognize certain aspects of the pathogenesis of inflammatory bowel diseases, but our ability to effectively treat food and pollen allergy remains limited. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot.ĬONCLUSION: 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.Ītopic diseases such as asthma, rhinitis, eczema, pollen allergies and food allergies have been increasing in most industrialized countries of the world during the last 20 years.
Recombinant allergen rAmb a 8(D106) was then successfully generated. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. 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. 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. 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.
Northern blot was conducted to confirm pollen sources of the gene. 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. 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. AIM: To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production.
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