RESUMO
Objective: To identify the dominant epitopes of α-8 giardin in Giardia lamblia, and analyze their immunoreactivity. Methods: Three dominant epitopes G1ï¼7-17 aaï¼, G2ï¼30-40 aaï¼ and G3ï¼296-306 aaï¼ were predicted for α-8 giardin by bioinformatic analysis using DNAstar, Biosun and CLUSTAL W softwares. Each epitope was conjugated to keyhole limpethemocyanin (KLH) and then used to immunize female Chinchilla rabbits via multi-pointï¼8-10 pointsï¼ subcutaneous injections on the backï¼0.5 mg conjugate/rabbit, 100 µl at each pointï¼. Then the rabbits were boostedï¼0.2 mg conjugate/rabbitï¼ on days 14, 21 and 28 after the first immunization. Blood was collected prior to each immunization, and carotid artery bloodï¼50 mlï¼ was collected on day 7 after the last immunization. The antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was determined with indirect ELISA. The reactivity of the three anti-sera with recominant giardin (rGiardin) was analyzed with Western blotting. Results: The concentration of the purified conjugates KLH-G1, KLH-G2, and KLH-G3 was 0.66, 0.95 and 0.25 mg/mlï¼ respectively. ELISA showed that the antibody titer for anti-KLH-G1, anti-KLH-G2 and anti-KLH-G3 sera was 1â¶12800, 1â¶51200, 1â¶51200, respectively. SDS-PAGE revealed a specific band at M(r) 36 000 for the three anti-sera. Western blotting showed that the three anti-sera all specifically recognized rGiardin. Conclusion: Immunization with the KLH-G1, KLH-G2, and KLH-G3 conjugates induced production of specific IgG antibody in rabbits, which can recognize rGiardin.
Assuntos
Giardia lamblia , Epitopos Imunodominantes , Animais , Formação de Anticorpos , Antígenos , Western Blotting , Proteínas do Citoesqueleto , Eletroforese em Gel de Poliacrilamida , Epitopos , Feminino , Imunização , Proteínas de Protozoários , CoelhosRESUMO
OBJECTIVE: To clone and express α8-giardin gene of Giardia lamblia, and analyze its immunoreactivity. METHODS: The open reading frame (ORF) of α8-giardin gene was amplified by PCR. The PCR product was cloned into prokaryotic expression vector pET-30a (+) with restriction enzymes EcoR I and Xho I . The recombinant vector pET30a (+)-α8-giardin was transformed into E. coli BL21 (DE3), and the positive clones were then selected. The constructed pET30 a (+)-α8-giardin was induced with IPTG for expression, and purified through Ni-affinity chromatography. The recombinant protein was examined by SDS-PAGE and Western blotting. RESULTS: The length of α8-giardin gene was 930 bp. PCR and restriction enzyme digestion analysis confirmed the construction of recombinant plasmid pET30a (+)-α8-giardin. SDS-PAGE and Western blotting analysis showed that the recombinant protein rGiardin (about M, 36,000) was expressed in E. coli as inclusion body protein, and reacted positively with anti-His tag antibody and rabbit anti-G. lamblia serum. CONCLUSION: The recombinant plasmid pET30a (+)-α8-giardin is constructed, and the purified rGiardin protein shows immunoreactivity.