Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells

Abstract

Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.

Allergens of <b><i>Blomia tropicalis</i></b>: An Overview of Recombinant Molecules

“…Likewise, rBlo t 6 produced in P. pastoris was more reactive than its counterpart produced in E. coli [40,46]. In another study, recombinant Blo t 11 produced in insect cells was clearly outperformed in IgE-binding assays by the natural counterpart [25]. These differences in IgE reactivity could be linked to the expression system used for recombinant production.…”

“…Using a glycosylated isoform of Blo t 12 as a control, Acevedo et al [60] were not able to detected glycan groups on natural Blo t 8 [62]. No glycan groups were found in recombinant Blo t 11 expressed in baculovirus, although the molecule has 5 predicted glycosylation sites [25,67]. No potential glycosylation sites were detected for Blo t 4 and Blo t 13 allergens [29,74].…”

“…Usually, recombinant allergens are produced in bacterial expression systems [59,61,72,79]. Although the majority of bacterium-based expression systems are able to produce biologically active and correctly folded molecules, for a few allergens a host that allows more elaborated posttranslational modifications is required [25,67,68,69]. In this respect, eukaryotic expression systems are able to perform posttranslational modifications such as disulfide bond formation, processing of signal sequences, lipid modifications, and glycosylation [80].…”

“…These developments were enabled by the application of recombinant DNA technology, which opened up new possibilities for the field of allergy, leading not only to the production of purified recombinant house dust mite allergens but also to the standardization of molecule-based preparations for diagnosis and allergen-specific immunotherapy [20]. Since the first data of a purified recombinant B. tropicalis allergen (rBlo t 5) published in 1995 by Arruda et al [21], several research groups have produced B. tropicalis allergens in recombinant form using different expression systems [12,22,23,24,25]. …”

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

“…The medium was incubated for 1h at 27⁰C and aspirated from the plate. An agarose solution at 5 mL, which included 50 μg/mL X-Gal (5bromo-4-chloroindol-3-yl β-D-galactopyranoside), was laid over the cells [44]. Plates were then incubated at 27⁰C for 7 to 10 days.…”