cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen
Giovanni DuroPaolo ColomboMaria Assunta CostaV. IzzoRossana PorcasiRenata Di FioreGiovanni LocorotondoMario G. MirisolaRoberta CocchiaraDomenico Geraci
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Abstract:
A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj‐allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11 344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj‐allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross‐inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10–14 kDa native allergens and preincubation of sera from Pj‐allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10–14 kDa native allergen region, suggesting that these two allergens contributed to the region.Keywords:
Cloning (programming)
In the present,plant genes cloning was the front and focus in the field of botany research.After the development of about twenty years,some methods for cloning plant genes were established,which included functional cloning,PCR amplifing cloning,transposon or T DNA tagging,postional cloning,differential hybridization and subtractive hybridization,mRNA differential display and artificial synthesis DNA cloning.In this paper,these techniques theory,advantages and disadvantages of each,as well as their application prospects in cloning plant genes were introduced and dicussed.
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A new peroxidase gene from Orychophragmus violaceus was cloned. The full-length cDNA of O.violaceus peroxidase gene (OvRCI, GenBank. Acc. No. AY428037) was 1220 bp and contained an 1128 bp open reading frame encoding a protein of 375 amino acids. Homology analysis and molecular modeling revealed that OvRCI strongly resembled other peroxidase genes. Quantitative real-time PCR analysis revealed that it was a constitutively salt-inducible gene and its transcript level was most abundant after 24 h treatment with 200 mmol.L-1 sodium chloride. Our studies suggested that OvRCI was a new member of the family of recently cloned peroxidase genes.
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Allergen-specific IgE production is the central event in the pathogenesis of atopic disorders and increases in specific IgE serum antibodies are an indicator of immediate hypersensitivity responses in humans and in animal models of allergy. Consequently, accurate and user-friendly methods are needed to measure serum levels of allergen-specific IgE. This review examines historical and recent developments in in vivo and in vitro methods for the detection of allergen-specific IgE in humans and in animal models. Routinely, in vitro methods such as enzyme-linked immunosorbant assays or radioallergosorbant tests and in vivo methods such as the skin prick test (SPT) for humans and the passive cutaneous anaphylaxis assay (PCA) used in animals are utilized to detect allergen-specific IgE. While in vivo assays are usually more accurate than in vitro assays since they provide a functional readout of IgE activity, they are relatively costly and require considerable expertise. On the other hand in vitro assays are limited by the fact that the amount of allergen-specific serum IgG exceeds IgE antibody by several orders of magnitude, resulting in competition for allergen binding. Consequently, methods that use allergen as a direct capture step are limited by the availability of free allergen binding sites for IgE. In order to circumvent this problem, in vitro methods usually require prior depletion of IgG or use high amounts of allergen in order to facilitate availability of free binding sites for IgE detection. Clearly, these approaches are limited for small sample volumes and allergens that are in short supply. New methods such as protein microarray could potentially overcome this problem by providing high allergen concentrations in a relatively small reaction volume. Currently, in vitro methods are rarely used in isolation for prognosis but are used primarily to complement the information obtained from in vivo assays. With the emergence of new technologies it is conceivable that in vitro assays may in the future replace in vivo assays, however until then in vivo assays remain the gold standard of allergen-specific IgE detection.
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A method for the detection of specific IgE in human serum is described, using a modified radioimmune assay termed the Inhibition Assay Technique (IAT) The technique is based on the ability of specific IgE to bind with allergen. The difference in total IgE before incubation with the allergen and after incubation with the allergen gives a relative concentration of specific IgE to that particular allergenic substance. The assay has been shown to correlate with both the intradermal skin test as well as the standard RAST now used to determine specific IgE.
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Abstract Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19‐residue toxin from Escherichia coli K‐12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this “detox cloning” system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC‐based cloning vectors are as reliable and amenable to high‐throughput cloning as commercially available systems, and have the advantage of being more time‐efficient and cost‐effective.
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Chalcone synthase is a key factor to flavonoids synthesis.AcCHS1 and AcCHS2,the two chalcone synthase genes,were cloned from onion.The open reading frame(ORF) of AcCHS1 gene contained 1 182 bp encoding a protein of 393 amino acids and the ORF of AcCHS2 gene contained 1 176 bp encoding a protein of 391 amino acids.Under the same conditions,the expression of AcCHS2 was higher than that of AcCHS1.
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In this book, a cloned novel modal has been reported that could be transfer to the plants to produce salt tolerant. Current, book includes the retrieval of gene sequence from database and designing of specific primer. Amplification of selected gene with the help of designed primer and cloning of amplified product through TA cloning strategies. Cloned gene was transformed in E. coli DH5α with the help of pRSET-A vector to express the gene. The results showed that the transformed E. coli tolerate up to certain salinity level. Current investigation clearly suggested an unpretentious approach to resolve the problems such as salt stress. This information would be highly useful for the understanding of molecular cloning of genes responsible for salt tolerance in natural environment.
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There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.
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