Cloning,Expression,purification Of Tyr P 28 From Tyrophagus Putrescentiae And Its Immunological Characteristics

Objecive To obtain the prokaryotic expression product for the group 28 allergen of Tyrophagus putrescentiae(Tyr p 28)and analyze its immunological characterization.Methods Dust samples were collected from the dormitory,hotel mattresses and flour mills from June to September.Under stereo microscope,single pregnant mite were picked and put into small bottles containing culture medium,which were then put in an artificial climate incubator set with the temperature of(25±1)? and the RH of 85±5%.Sixty days later,the mites were identified under inverted microscope,and the Tyrophagus putrescentiae will be cultured on a large scale.The total RNA was isolated from Tyrophagus putrescentiae and used for RT-PCR amplification of the gene fragment encoding Tyr p 28 with the primers designed and synthesized according to GenBank Accession Number KX060611.The gene Tyr p 28 was then cloned into expression vector pET28a(+).After correct sequencing,the plasmid pET28a(+)-Tyr28 was transformed into E.coli BL21(DE3)T1R for expression with induction agents of isopropy-?-D-thiogalactoside(IPTG),which was identified with SDS-PAGE and Western blot.The expression product was submitted for purification with affinity chromatography,and quantified by microultraviolet-visible spectrophotometer.The recombinant protein rTyr p28 was tested by western blot with Tyrophagus putrescentiae-allergic dermatitis patients sera as the primary antibody.Results The mites Tyrophagus putrescentiae were cultured in laboratory successfully.The plasmids pET28a(+)-Tyr p 28 were constructed,transformed into E.coli BL21(DE3)T1R and expressed successfully.SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and sera IgE.The IgE-binding rate of recombinant allergen Tyr p 28 was 38.1%(8/21)with sera from Tyrophagus putrescentiae-allergic dermatitis patients.Conclusion The recombinant protein rTyr p 28 was obtained in the present study.The present study provide a good foundation for clinical diagnosis,treatment and further experimental studies of the dust mite allergy disease.