We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, ...We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.展开更多
A new strain of Lactococcus lactis producing D-tagatose was isolated and identified.Its L-arabinose isomerase coding gene was cloned and expressed in Escherichia coli BL21(DE3).The optimal temperature and pH of the pu...A new strain of Lactococcus lactis producing D-tagatose was isolated and identified.Its L-arabinose isomerase coding gene was cloned and expressed in Escherichia coli BL21(DE3).The optimal temperature and pH of the purified enzyme were 50◦C and 8.0.To produce D-tagatose from lactose,β-D-galactosidases from Lc.Lactis,Lactiplantibacillus plantarum,and Streptococcus thermophilus were further incorporated into E.coli by two strategies.Theseβ-D-galactosidases were fused to L-arabinose isomerase coupled with a peptide linker(GGGGS)3.Meanwhile,they were co-expressed with L-arabinose isomerase using pETDuet-1 vector.Among these recombinant strains,the cell co-expressing L-arabinose isomerase and S.thermophilusβ-D-galactosidase showed maximal activity.SDS-PAGE results confirmed that exogenous enzymes had the maximum soluble expression level in this strain.At the optimal condition,the conversion rate of D-tagatose from 300 g/L lactose achieved to 42.4%,and the volumetric productivity reached 4.28 g/L/h at 15 h.This research established a highly efficient biotransformation system to produce D-tagatose from lactose.展开更多
文摘We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.
基金supported by Natural Science Foundation of Shandong Province(ZR2021QC160)Key Research and Development Program of Shandong Province(2020CXGC010602)Science and Technology Support Plan for Young People in Colleges and Universities of Shandong Province(2020KJE005).
文摘A new strain of Lactococcus lactis producing D-tagatose was isolated and identified.Its L-arabinose isomerase coding gene was cloned and expressed in Escherichia coli BL21(DE3).The optimal temperature and pH of the purified enzyme were 50◦C and 8.0.To produce D-tagatose from lactose,β-D-galactosidases from Lc.Lactis,Lactiplantibacillus plantarum,and Streptococcus thermophilus were further incorporated into E.coli by two strategies.Theseβ-D-galactosidases were fused to L-arabinose isomerase coupled with a peptide linker(GGGGS)3.Meanwhile,they were co-expressed with L-arabinose isomerase using pETDuet-1 vector.Among these recombinant strains,the cell co-expressing L-arabinose isomerase and S.thermophilusβ-D-galactosidase showed maximal activity.SDS-PAGE results confirmed that exogenous enzymes had the maximum soluble expression level in this strain.At the optimal condition,the conversion rate of D-tagatose from 300 g/L lactose achieved to 42.4%,and the volumetric productivity reached 4.28 g/L/h at 15 h.This research established a highly efficient biotransformation system to produce D-tagatose from lactose.