Objective: To clone, express, purify the Per a 4 gene encoding an allergen of Periplaneta Americana and prepare monoclonal antibodies against the recombinant allergen.
Methods: The total RNA was extracted from P. Americana, and the target gene was amplified by RT-PCR and cloned into pMD18-T vector. After being confirmed by nucleotide sequencing, the gene was then inserted into pGEX-3X to construct the express vector pGEX-3X-Per a 4. Further, the pGEX-3X-Per a 4 was transformed into E. coli BL21 (DE3), and induced for expression by IPTG. By affinity chromatography, the recombinant allergen was purified and identified by SDS-PAGE and Western blotting. The BALB/c mouse was immunized with the recombinant allergen to prepare the specific monoclonal antibodies, which was then identified by co-immunoprecipitatin and western blotting.
Results: The full-length cDNA encoding Per a 4 of P. Americana was obtained with 552 bp in length, which had 99.4% similarity with the reference sequence (GenBank AY792948). The constructed vector pGEX-3X-Per a 4 was transformed in E. coli BL21 (DE3), expressed with the induction of IPTG. By SDS-PAGE, a band of about 49 KD was present. Further, the western-blotting showed that the prepared monoclonal antibodies can bind the serum antibodies in patients allergic to P. Americana.
Conclusions: Both the recombinant allergen Per a 4 and its monoclonal antibodies were obtained.
Keywords: Per a 4; Periplaneta Americana; allergens; monoclonal antibody.
© 2018 by the Association of Clinical Scientists, Inc.