Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Sehistosorna japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The V_H and V_L genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F' induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Westem-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.

Abstract OBJECTIVE To study the effect of berberine(BBR) on autophagy of human breast cancer HCC1937 cells under hypoxia condition. METHODS Cultured human breast cancer HCC1937 cells, CCK-8 method was used to determine the effects of different concentrations of BBR(0, 5, 10, 20, 40, 80, 160 μmol·L-1) on cell viability under normoxia and hypoxia conditions, and select the drug concentration for further experiments. Cultured HCC1937 cells were randomly divided into 4 groups: control group, 20 μmol·L-1 BBR group, hypoxia group, hypoxia+20 μmol·L-1 BBR group. LIVE/DEAD cell viability/cytotoxicity kits were used to measure the cell death rate. The expressions of autophagy related proteins Beclin1, LC3 and P62 in each group were determined by Western blotting. The cells were infected with mCherry-GFP-LC3 adenovirus, and the number of autophagosomes and autophagolysosomes in each group were counted by laser confocal microscopy to determine the effect of BBR on the autophagy flow of HCC1937 cells. RESULTS BBR decreased the cell viability of human breast cancer HCC1937 cells in a concentration-dependent manner. After hypoxia treatment, the cell death rate of HCC1937 cells was not significantly changed, and the intracellular Beclin1, LC3-II and LC3-II/LC3-I ratio were significantly increased, while P62 without significant changes, and the autophagy flow was increased. BBR significantly increased cell death rate, decreased Beclin1 and LC3II/LC3-I ratio, increased intracellular P62, significantly reduced the number of autophagosomes and autophagolysosomes, and inhibited the formation and clearance of autophagosomes under both normal and hypoxia conditions. CONCLUSION BBR increases the death rate of human breast cancer HCC1937 cells under hypoxia condition, and its effect is related to the inhibitory effect of berberine on autophagy under hypoxia condition.