PRODUCTION OF RECOMBINANT GROUP 10 ALLERGENS OF DERMATOPHAGOIDES PTERONYSSINUS AND TYROPHAGUS PUTRESCENTIAE MITES AND MONOCLONAL ANTIBODIES AGAINST THEM FOR THE STUDY OF MITE ALLERGY

Abstract

Mites are eight-legged acari that are classified under the class of Arachnida. Mites are diverse groups of invertebrate that can survive in different habitats. Domestic mites include the house dust mites (HDM) and storage mites that are associated with the home environment. These are the mites of medical and economic importance. Inhalation of dead or live mites, their faecal matter and other byproducts from our living environment can trigger asthma, rhinitis and contact dermatitis. The two species of mites under this study, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae, belong to the group of HDM and storage mites respectively. HDM infestation occurs at homes, including in carpets, mattresses and bedding, clothing, stored food products, and house hold pets. HDM is a major causative agent for asthma and rhinitis in Malaysia. Storage mites represent one of the occupational hazards for farmers, field hands, mill workers, warehouse operators and other food industrial workers. Group 10 allergen of mite is a muscle protein, tropomyosin, which has received attention due to its cross-reactivity with some allergens from invertebrates, particularly in seafood, responsible for severe systemic anaphylaxis. The objectives of this study were to produce recombinant Group 10 allergens of D. pteronyssinus and T. putrescentiae and to assess their IgE binding reactivity with human sera; to produce monoclonal antibodies against these recombinant allergens and to apply them in the localisation of inhaled mite Group 10 allergens in respiratory system of animal models. Two species of mites, D. pteronyssinus and T. putrescentiae were cultured and harvested. The mites were homogenised and the crude mite proteins were used to immunise the mice for the production of anti-mite polyclonal antibodies. Total RNA of mites was isolated and cDNA was constructed. Specific primers were designed for the amplification of group 10 allergen genes of both species of mite (Der p 10 and Tyr p 10) through polymerase chain reaction (PCR). The PCR produced a 855 bp band, which is in accordance with the reported molecular weight of mite tropomyosin in previous studies. DNA sequencing of the PCR products further confirmed that they are the group 10 allergen genes. The PCR products were purified and digested with restriction enzymes before they were cloned into the expression vector pRSET C. The recombinant plasmids (pRSET C/Der p 10 and pRSET C/Tyr p 10) were purified and transformed into the expression host E. coli, BL21. Colony PCR was carried out to screen and to select the colonies of BL21 that carried the recombinant plasmids. The selected positive BL21 clones were subjected to recombinant allergen protein expression under the induction of IPTG. The recombinant allergens were screened in western blot by using anti-His antibody and sera of immunised mice. A protein band of about 38 kDa which represent the group 10 mite allergen was detected. The recombinant allergens were purified with nickel-nitrilotriacetic acid (Ni-NTA) resin affinity column. Most of the unwanted bacterial proteins were removed throughout the purification process leaving mainly the recombinant allergen protein (38 kDa) in the elution fraction. A panel of 44 human sera from allergic and normal human subjects was used to assess the IgE binding reactivity of the recombinant mite tropomyosin by dot blot immunoassay. The results showed 4.5% (2 / 44) and 2.3% (1 / 44) positive reaction to recombinant Der p 10 and recombinant Tyr p 10 respectively. The purified recombinant mite allergens were used to immunise the mice for the production of monoclonal antibodies against group 10 allergen. The spleens of the mice were removed and splenocytes were fused with NS1 myeloma cells. The hybridoma cells were screened for the presence of anti-mite tropomyosin specific antibody using dot blot immunoassay and western blot. One positive hybridoma clone for each species of mites was selected for the study. The selected positive hybridoma clones were DP10-F11 (D. pteronyssinus) and TP10-C5 (T. putrescentiae). The result of antibody isotyping showed that both of them were IgG1 with κ light chains. DP10-F11 antibody was found to be cross-reactive against Tyrophagus putrescentiae (68%), Glycycometus malaysiensis (50%), Aleuroglyphus ovatus (51%), Blomia tropicalis (62%), D. farinae (102%) and shrimp (56%). TP10-C5 antibody demonstrated cross-reactivity with Dermatophagoides pteronyssinus (63%), Blomia tropicalis (59%), Dermatophagoides farinae (82%) and shrimp (58%). However, both of these antibodies did not cross-react with E. coli (< 50%). The hybridoma cells of DP10-F11 and TP10-C5 were intraperitoneally injected into the pristine-primed BALB/c mice and allowed to propagate inside them for the production of ascites fluid. The ascites fluids collected from mice were purified using the HiTrap Protein G HP column and fractioned protein liquid chromatography (FPLC). Both the purified monoclonal antibodies from ascites fluids showed positive reactivity to the recombinant mite tropomyosin in dot blot immunoassay and indirect ELISA. Female inbred Balb/c mice (n=6) were intranasally challenged with purified recombinant Der p 10 and recombinant Tyr p 10 for 10 consecutive days. Positive and negative control groups (n=6 each) were challenged with ovalbumin and PBS respectively. The mice from each group were re-challenged on day 17 for 3 consecutive days. The mice were sacrificed via cardiac puncture on the next day after re-challenge. Anti-mite tropomyosin polyclonal antibodies were detected in the sera of mice intranasally challenged with recombinant allergens. Histopathological changes such as infiltration of inflammatory cells and hypertrophy of airway epithelial cells were observed in the lung and trachea of mice challenged with recombinant allergens. The results of immunohistochemistry staining showed that both DP10-F11 and TP10-C5 antibodies demonstrated positive immunoreactivity at the alveolar epithelium in the lung tissues and fibrocartilaginous areas of the trachea. The recombinant group 10 allergens (tropomyosin) of D. pteronyssinus and T. putrescentiae were successfully produced in this study. Both of these recombinant allergens demonstrated their allergenicity through IgE binding reactivity with human sera. Monoclonal antibodies against recombinant group 10 allergens of D. pteronyssinus and T. putrescentiae were produced and were able to localize the inhaled allergens of D. pteronyssinus and T. putrescentiae in the respiratory system of mice. Both the research hypotheses of this study were accepted and all the objectives were achieved.