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1.
Article in Chinese | WPRIM | ID: wpr-380123

ABSTRACT

Objective To develop mycobacterial inducible expression vectors which permit to overexpress Mycobacterium tuberculosis (Mtb) immunodominant antigen, and to analyze its immunogenicity after purification by affinity chromatography. Methods The regulatory region of M. smegmatis (Ms) acet-amidase(pACE) was obtained by PCR amplification, and was used as promoter to construct the mycobacteri-al inducible expression vectors, pMF series. The coding gene of Mtb chimeric antigen Ag856A2 which is a recombinant Ag85A with 2 copies of ESAT-6 inserted in its Acc Ⅰ site and showed excellent immunogenicity in the animal experiments we described previously, was cloned into the pMF vector series, and was induced to express by the addition of acetamide. The recombinant protein expressed in the Ms was purified by the Ni~(2+)-NTA affinity chromatography, the resulted homologous recombinant antigen was added into the spleen cells separated from BCG vaccinated mice, and the immunogenicity was analyzed by the IFN-γ ELISPOT as-say. Results The mycobacterial inducible expression vectors, pMF series was constructed successfully, target antigen could be. induced to express in the Ms by the addition of 0.02% acetamide, and could be puri-fied by the Ni~(2+) -NTA affinity chromatography due to the addition of 6×His tag in the vector pMF406. Fur-thermore, the mycobactefial homologous antigen could induce more IFN-γ secretion than the heterogonous one. Conclusion The mycobacterial inducible expression system based on the regulatory region of Ms acet-amidase as promoter could permit the Mtb target antigen of interest overexpression and purification, and the immunogenicity of the homologous antigen from Ms is better than that of be expressed from E. coli, which may be more potential for immunological detection of tuberculosis.

2.
Article in Chinese | WPRIM | ID: wpr-686149

ABSTRACT

Aspergillus oryzae is an important microorganism,which was widely used in the fields of food,brewing,pharmacy and fermentation industry etc and was termed as a generally regarded as safe(GRAS) organism by the United States Food and Drug Administration(FDA).The strategies for promoting the expression of homologous and heterologous proteins in Aspergillus oryzae were reviewed.Which included the usage of strong promoters,multicopies of coding genes,optimization of culture medium and overexpression of the hemoglobin domains(HBD) etc.Heterologous proteins were usually exhibited low expression in Aspergillus oryzae due to the degradation by the proteinases from host.Therefore,development of proteinase auxotrophic stains of Aspergillus oryzae as host is neccessary.In addition,fusion of the heterologous protein with a highly secreted protein of Aspergillus oryzae was an alternative way to prompt the expression of heterologous protein in Aspergillus oryzae.

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