RESUMO
OBJECTIVE: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity. METHODS: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. RESULTS: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. CONCLUSION: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.
Assuntos
Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/biossíntese , Toxoplasma/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Escherichia coli/genética , Feminino , Expressão Gênica , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Toxoplasma/genéticaRESUMO
OBJECTIVE: To make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E. coli and study the antigenicity of the expressed product. METHODS: The SAG2 gene fragment of T. gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E. coli DH5alpha. After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E. coli DH5alpha. The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E. coli BL21 (DE3) and induced for expression. The expressed product was studied by SDS-PAGE and Western blot. RESULTS: 502 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about Mr 19,000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSION: The plasmid pET23a-SAG2 was constructed and a high efficiency expression of the SAG2 gene from T. gondii RH strain was made. The expressed product shows a specific antigenicity.