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 coimmunoprecipitatin 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 westernblotting 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.