Detection of Periplaneta americana sIgE with Chemiluminescent Immunoassay(CLIA) / 中国寄生虫学与寄生虫病杂志

Crude extract of Periplaneta americana was prepared by liquid nitrogen grinding.After being purified with DEAE Sephadex A-50 ion exchange chromatography,the protein content of the extract was determined and the extract solution was prepared at gradient concentrations.The crude extract and purified allergen at different concentrations were dotted respectively on nitrocellulose(NC) membrane.Patient serum,bio-IgE,sa-HRP,luminal regents were added to the membrane.The chemiluminescence was displayed by exposing to X-film.The result revealed that the minimum protein content of crude Periplaneta americana extract detected by CLIA is 0.87?g/ml,with 90% accordance to skin test positive patients,and 100% accordance to those with negative skin test and ELISA detection.

Cloning, Expression and Purification of Allergen Arginine Kinase from Periplaneta americana and its Allergic Activity / 中国寄生虫学与寄生虫病杂志

Objective To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity. Method The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindⅢ. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA). Result The cloned cDNA ORF sequence (Accession no. EU429466) contained 1 068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45 000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA. Conclusion The recombinant cockroach arginine kinase has been obtained with proper allergenicity.

Cloning,Expression and Purification of Dust Mite Allergen Der f 3 and Identification of its Allergic Activity / 中国寄生虫学与寄生虫病杂志

Objective To clone,express and identify Der f 3 gene.Methods Live mites were collected from southern China region,identified as Dermatophagoides farinae,and cultured.The total RNA was extracted.The Der f 3 gene fragment was amplified by RT-PCR and sequenced.The Der f 3 gene fragment encoding a serine protease mature peptide was sub-cloned into the expression vector pET-His.The recombinant pET-Der f 3 plasmid was inserted into E.coli BL21 and induced to express Der f 3 coding protein by IPTG.The recombinant Der f 3 with 6 his-tag was then purified by chelating resin and its allergic activity was identified by Western blotting.Results The Der f 3 gene fragment with 840 bases was determined.Its sequence homology with the published one(GenBank No.D63858) was 99.5% at nucleotide level.It was sub-cloned into expressing vector pET-His and the recombinant allergen rDer f 3 was highly expressed in E.coli BL21(DE3) under induction of IPTG,and purified by 6-His-tag purification system.Using Western blotting method,the allergic activity of the purified recombinant allergen was identified as its affinity to IgE antibodies from the mite-allergic patient sera.Conclusion Der f 3 gene has been successfully cloned and its prokaryotic expression vector is constructed.