American Cockroach Nymphs Change To The Original Cr Pi Expression, Purification And Immunological Characteristics Of Identification

Background: Allergic diseases, such as asthma, allergic rhitis etc, affect about 10-30% of world population and the incidence is still increasing rapidly. Of a great variety of allergens, cockroach is considered one of the most important inhaled allergens,and Periplaneta Americana is the dominant cockroach spiece in China . To date, crude Periplaneta Americana extract is commonly used for specific diagnosis in vivo or in vitro and immunotherapy of cockroach allergic disease, but these crude extract often contains large number of non-allergic components and is hard to be standardized. So, researches on recombinant allergens are of great interest in recent years. Objective: To clone, express and characterize the major Periplaneta Americana allergen Cr PI. Methods: Using the Cr PI clone from the λEXcell library as a template, the cDNA fragments were first generated by PCR techniques and were then ligated into pUCm-T. After confirmed by DNA sequencing, the cDNA encoding Cr PI allergen was subcloned into PGEX-5X-1, resulting in a GST-Cr PI fusion gene construction (pGEX-5X-1/Cr PI). This plasmid was transformed into and expressed by the Escherichia coli strain BL21. Most of the expressed fusion protein was insoluble. After intensive washing, the proteins of interest from the inclusion bodies were isolated and dissolved in 6 mol/L guanidine hydrochloride (Gdn-HCl). Refolding was done by rapid dilution method. The effect of protein concentration and L-arginine concentration on aggregation of diluted inclusion bodies was evaluated. The renatured fusion proteins were purified by Affinity chromatography with Glutathione Sepharose 4B and subjected to Western blotting using sera from subjects allergic to cockroach. The renatured fusion proteins was cleaved by Xa and then subjected to size exclusion chromatography to separate the GST tag and the Cr PI allergen. After the targeted allergens being in-gel digested by trypsin, the peptides obtained were analyzed by inline HPLC/ESI-MS/MS, the experimental peptide patterns were searched in the Mascot database.Results: Following the IPTG induction, the cockroach major allergen Cr PI was overexpressed in E.coli BL21 and the expressed fusion proteins were increased with the time going. The solubility analysis showed that most of the recombinant allergens were found in the insoluble fraction (inclusion body) of the cell lysate. Optimal refolding results were achieved when the concentration of refolding proteins was below 320μg/ml and that of L-arginine was about 0.5 mol/L. The fusion proteins were highly purified through affinity chromatography with Glutathione Sepharose 4B。The recombinant Cr PI was shown by Western blotting to possess good IgE-binding capacity. After Xa cleavage of the GST tag, the Cr PI allergen was purified by size exclusion chromatography. Mascot search results show that the scores for P.Americana Cr PI allergen are 412(Protein scores greater than 73 are significant),Conclusions: The cockroach major allergen Cr PI was overexpressed in E.coli and highly purified. Western blotting assay indicated the recombinant allergen had good IgE-binding activity. This study has laid a base for the specific diagnosis and further experimental studies of cockroach allergic disease.