Construction of recombinant plasmids Der f 6/pET32a (+) and its expression, purification,IgE-binding reactivity / 中国免疫学杂志

ABSTRACT

Objective:To obtain the prokaryotic expression product for the group 6 allergen of Dermatophagoides farine ( Der f 6) and detect its IgE-binding rates with sera from asthmatic children. Methods: By enzyme digestion of pET28a (+)-Der f 6 with BamHⅠ plus XhoⅠ,the target gene Der f 6 was obtained and linked into the vector pET32a (+) to construct the recombinant plasmid pET32a(+)-Der f 6, which was then transfected into E. coli BL21 cells for expression, induced with isopropyl-β-D-thiogalactoside ( IPTG) ,purified by affinity chromatography and identified by SDS-PAGE,Western blot and AMLDI-TOF,and tested by ELISA for IgE reactivity with sera from asthmatic children. Results:The plasmids pET32a(+)-Der f 6 were constructed,transformed into E. coli BL21 and expressed successfully. SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and the His-tag in the plasmids,and MALDI-TOF/TOF identified the identical structure to the allergen Der f 6. Using the ELISA method developed with the recombinant proteins as coating antigen,the positive rate was 41. 3% (19/46) in asthmatic children allergic to dust mite. Conclusion: The plasmids pET32a (+)-Der f 6 were constructed successfully,expressed in E. coli BL 21 (DE3). The recombinant fusion protein has a good reactivity with sera from asthmatic children.