RESUMO
OBJECTIVE: To clone and express the aegyptin-like protein (alALP) encoding gene from Aedes albopictus salivary gland, and analyze its antigenicity. METHODS: The homology, secondary structure and antigen peptides of alALP and aegyptin protein (GenBank No. ABF18122.1) was analyzed by bioinformatics software tools. Total RNA was extracted from Ae. albopictus salivary gland. The coding region of alALP (GenBank No. AY826121) was amplified by PCR. RT-PCR product was digested with restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-alALP plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant soluble GST-alALP fusion protein was purified with Glutathione Sepharose 4B. The expression product was analyzed by SDS-PAGE and Western blotting. Mice were immunized each with 60 microg purified GST-alALP at every 2 weeks for 3 times, and mouse anti-GST-alALP serum was prepared. Western blotting assay with mice anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites was used to analyze its antigenicity. RESULTS: Bioinformatics prediction results showed that alALP and aegyptin had 65.58% homology with a similar secondary structure, and a conservative polypeptide. The product of RT-PCR was 762 bp. The recombinant plasmid pGEX-6P-1-alALP was confirmed by double restriction enzyme digestion, PCR and sequencing. SDS-PAGE and Western blotting analysis showed that the bacteria containing recombinant plasmid pGEX-6p-1-alALP expressed a soluble recombinant fusion protein (M(r) 56 000) after being induced with IPTG. Western blotting analysis revealed that GST-alALP protein was recognized by mouse anti-GST-alALP serum and serum of mice ex- posed to Ae. albopictus bites. CONCLUSION: Mature peptide gene of alALP can be expressed in prokaryotic expression system, and the recombinant protein shows antigenicity.
