Reports of Biochemistry and Molecular Biology
Volume:1 Issue: 1, Oct 2012

The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb''s-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide. Therefore, was designed a study for antimicrobial-resistance determination in this strain. Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used. Results & We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Allergy is a clinical disorder affecting humans worldwide. Allergenic extracts prepared from natural source materials remain heterogeneous in composition and content, but are regularly used for diagnosis and immunotherapy. Recombinant allergens are suitable candidates to use in place of natural allergens; however, the recombinant allergens should be assessed and compared with the natural ones. Cuc m 2 (profilin), one of the most important allergens of melon (Cucumis melo), has been cloned and was expressed in Escherichia coli (E. coli). We aimed to evaluate the validity of recombinant Cuc m 2 (rCuc m 2) in the diagnosis of melon allergy and investigate whether rCuc m 2 could be used as a replacement for natural Cuc m 2 (nCuc m 2). nCuc m 2 was purified by immuno-affinity chromatography and rCuc m 2 was purified by metal-affinity chromatography. SDS-PAGE and western blotting were carried out to evaluate the purification methods. Skin prick tests (SPT), and enzyme immunoassays to determine specific IgE, were performed with the natural and recombinant purified allergens on 53 patients with melon allergy. rCuc m 2 elicited no significantly different responses in skin compared with nCuc m 2. All patients'' sera showed similar ODs in ELISAs with natural and recombinant profilin. rCuc m 2 evoked strong immuno-reactivity equivalent to nCuc m 2, and has potential for diagnosis of melon allergy.

DNA immunization with plasmid DNA encoding bacterial, viral, parasitic, and tumor antigens has been reported to trigger protective immunity. The use of plasmid DNA vaccinations against many diseases has produced promising results in animal and human clinical trials; however, safety concerns about the use of DNA vaccines exist, such as the possibility of integration into the host genome, and elicitation of adverse immune responses. In this study, we examined the potential integration and bio-distribution of pcDNA3.1+PA, a new vaccine candidate with GenBank accession # EF550208, encoding the PA63 gene, in reproductive organs of mice; ovaries and uterus in female, and testis in male. Animals of both sexes were injected intramuscularly with pcDNA3.1+PA. Host genome integration and tissue distribution were examined using PCR and RT-PCR techniques, two times monthly for six months. RT-PCR confirmed that pcDNA3.1+PA was not integrated into the host genome and did not enter reproductive organs. This finding has important implications for the use of pcDNA3.1+PA plasmid as a vaccine and opens new perspectives in the DNA vaccine area.

Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.

Recently, reports have indicated a role for the membrane form of Toll-like Receptor 2 (TLR2) in asthma pathogenesis. In this study we examined soluble TLR2 levels in serum and sputum of asthmatic and healthy subjects. Serum and sputum samples were obtained from 33 asthmatic and 19 healthy subjects. The asthmatics were classified into four groups according to the Global Initiative for Asthma. A sandwich ELISA was developed to measure soluble TLR2 (sTLR2) in serum and sputum. TLR2 mRNA expression was determined by semi-quantitative RT-PCR of all sputum samples. The mean sTLR2 levels from serum and sputum of asthmatics were significantly lower than healthy subjects. Moreover, sTLR2 concentration decreased concomitantly with asthma severity. The differences observed, however, were not statistically significant. TLR2/GAPDH mRNA of sputum leukocytes was also significantly lower in asthmatics than in healthy subjects. This study demonstrated for the first time that sTLR2 levels are lower in serum and sputum samples from asthmatic than from healthy subjects, and this could be an indicator of TLR2 expression. We also found that sTLR2 concentration in serum decreased concomitantly with an increase of asthma severity clinical score.

Despite many efforts, the etiology of autism remains unknown. Food allergy has been suggested as a pathogenic factor in Autism Spectrum Disorder (ASD). Our aim in this study was to determine whether food allergy could be considered as a risk factor for autistic children. Thirty-nine autistic children were examined by the skin prick test (SPT), and total serum IgE was evaluated by ELISA. SPTs were performed for egg whites, oranges, peanuts, tomatoes, tuna fish, walnuts, aubergines, melons, grapes, and cow milk. Parents and teachers were then asked to exclude these items from the childrens’diets for six months. After the treatment period, the autistic children who tested positive for food allergies were re-assessed by a standard questionnaire to obtain further information about their medical histories. Three of the study’s 39 autistic children (7.7%) tested positive on the SPT. Total serum IgE levels were elevated in 56.4% of the subjects (mean=164±24.5, cut-off >155 IU/ml). The results showed a decreased mean in the childrens’autistic behaviors on the Children Autism Rating Scale (CARS) after both eight weeks and six months; however, this decrease was not statistically significant. Food allergy may play a role in the pathophysiology of autism. We conclude that avoidance of certain foods benefits the behavior of autistic children.