Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells.
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.
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Singapore.
Audrey S M
AS
Teo
John D A
JD
Ramos
Bee Wah
BW
Lee
Nge
N
Cheong
Kaw Yan
KY
Chua
D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology; D052179:Antigens, Plant; D016367:Baculoviridae / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007313:Insecta / Q000166:cytology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D012846:Singapore; D014335:Tropomyosin / Q000096:biosynthesis* / Q000235:genetics
D000485:Allergens; D039741:Antigens, Dermatophagoides; D052179:Antigens, Plant; C448821:Blo t 11 allergen; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E