ChongqingMedical University

China

ChongqingMedical University

China
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He Z.-L.,ChongqingMedical University | Jiang K.-A.,ChongqingMedical University | Song F.-Z.,ChongqingMedical University | Ma Y.-P.,ChongqingMedical University
Chinese Journal of Biologicals | Year: 2012

Objective: To express and purify the FasA subunit of fimbrial protein of enterotoxigenic Escherichia coli (ETEC) and determine its immunogenicity. Methods: The fasA gene without signal peptide at 5′-terminus was cloned into the prokaryotic expression vector pQE-30, and the constructed recombinant plasmid pQE-30-fasA was transformed to E. coli M15 and induced with 0.5 mmol/L IPTG. The expressed fusion protein 6 x His-FasA was purified by protein purification kit with Ni-Agarose His tag and refolded. BALB/c mice were immunized with the fusion protein by subcutaneous injection at several sites, at a dosage of 100 μg, for 3 times each at an interval of 2 weeks, of which the sera were collected 10 d after the last immunization and determined for titer by ELISA. Results: PCR, restriction analysis and sequencing proved that recombinant plasmid pQE-30-fas4 was constructed correctly. The expressed fusion protein 6 x His-FasA, with a relative molecular mass of about 18 500, mainly existed in a form of inclusion body, contained 30% of total somatic protein, reached a purity of 95% and a concentration of 0.6 mg/ml, and induced a serum antibody titer of 1 : 125 000 in mice. Conclusion: Fusion protein 6 x FasA was successfully expressed in E. coli M15 and showed good immunogenicity after purification, which laid a foundation of further preparation of Bifidobacterium-based novel oral ETEC sub-unit vaccine.

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