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Objective@#To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy.@*Methods@#Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal.@*Results@#The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence.@*Conclusions@#The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.
ABSTRACT
Objective To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy. Methods Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal. Results The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence. Conclusions The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.
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Isomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.
Subject(s)
Oligosaccharides , Starch , Yarrowia , beta-AmylaseABSTRACT
The bioinformatics software was used to predict the B cell and T cell epitopes of Toxoplasma gondii microneme 16 (TgMIC16) followed by epitopes display on the yeast of Saccharomyces cerevisiae.B and T cell epitopes of TgMIC16 were predicted by DNAStar and IEDB,and software of SYFPEITHI and BIMAS,respectively.According to the predicted results,the antigenic epitope region was expressed with pCTCON2/EBY100 display system.The expression protein was detected by indirect immunofluorescence (IFA) and flow cytometry.Results showed that there were potential B/T cell epitopes in TgMIC16 and concentrated in the aa343-625 region.The epitope region was successfully displayed on the surface of yeast cells,and the optimal induction time was 24 hours.The study would lay the foundation for the development and efficacy evaluation of the yeast carrier vaccine in the further research.
ABSTRACT
The bioinformatics software was used to predict the B cell and T cell epitopes of Toxoplasma gondii microneme 16 (TgMIC16) followed by epitopes display on the yeast of Saccharomyces cerevisiae.B and T cell epitopes of TgMIC16 were predicted by DNAStar and IEDB,and software of SYFPEITHI and BIMAS,respectively.According to the predicted results,the antigenic epitope region was expressed with pCTCON2/EBY100 display system.The expression protein was detected by indirect immunofluorescence (IFA) and flow cytometry.Results showed that there were potential B/T cell epitopes in TgMIC16 and concentrated in the aa343-625 region.The epitope region was successfully displayed on the surface of yeast cells,and the optimal induction time was 24 hours.The study would lay the foundation for the development and efficacy evaluation of the yeast carrier vaccine in the further research.
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Objective:To analyze the immunogenicity of the extracellular region of Δ42PD1.Methods: Six fragments ofΔ42PD1 extracellular region-encoding sequence were amplified by PCR, and were cloned into pCTCON2 vector, a yeast surface displaying vector.Yeast cells were transfected with Δ42PD1 fragment-carrying plasmids, then yeast cells were spread on SDCAA plates.Single cell clones were selected and cultured in SGCAA media to induce expression of the target genes.Mouse anti-humanΔ42PD1 anti-serum were generated by immunization of BALB/c mice via intramuscular injection ofΔ42PD1-carrying plasmid plus in-situ electroporation.The binding of anti-serum with yeast cells surface-displaying Δ42PD1 fragments were analyzed using flowcytometry.Results:Nucleotide sequences analysis indicated that the amplified six fragments ofΔ42PD1 sequence length were 110 bp,and the isolated sequence ofΔ42PD1 fragments were 100%homology with PD1 gene previously registered in GenBank.Results from flowcytometry showed that among the six fragments of Δ42PD1 displaying on the surface of yeast cells,F3 and F2 profoundly boundΔ42PD1-specific polyclonal antibodies.Conclusion:F3 and F2 ofΔ42PD1 is an immunogenic dominant region,which pave the way for generation of Δ42PD1-specific monoclonal antibody and epitope mapping.
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ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.