To determine the inhibition of IL-13 by recombinant sIL-13Rα2 in NIH-3T3 fibroblast cells for its potential therapeutic value in hepatic fibrosis caused by Schistosoma japanicum in mice . IL-13 and sIL-13Rα2 from liver of BALB/c mice infected with S.japonicum at different infection time (weeks 0,6,8,10 and 12) were analyzed by ELISA and RT-PCR. The recombinant sIL-13Rα2 expression plasmidwas constructed, followed by transfection into NIH-3T3 fibroblast cells. TypeⅠcollagen produced by NIH-3T3 cells were examined by RT-PCR and Western blotting. It was demonstrated that the expression of IL-13 increased gradually after infection, reached peak density (16.1586 pg/mL)at week 8 and then reduced but was still higher than the level of control mice(3.4146 pg/mL;P =0.017 ). The secretion of sIL-13R α2 reached to its peak 10 weeks after infection(4827.426 pg/mL)and then reduced slowly but still higher than normal(4057.112 pg/mL; P=0.021). Meanwhile, the changes in mRNA level of IL-13 and sIL-13R α2 were coincided with that examined by ELISA. Both IL-13 and sIL-13Rα2 reached their peak density (P=0.033) at week 8 and 10 (P=0.025) respectively, and they were followed by a slower degree of decrease. The sIL-13Rα2 could significantly inhibit the effect of IL-13 on NIH-3T3 fibroblast cells, showing decreased mRNA level(P =0.012)and protein level of typeⅠcollagen compared with normal groups(P =0.031). It is concluded that the sIL-13Rα2 can inhibit the effect of IL-13 on NIH-3T3 fibroblast cells which leads to a reduced production of typeⅠcollagen, demonstrating its potential therapeutic value in hepatic fibrosis of schistosomiasis.

Objective:To construct an in vitro reconstitution system for inverse autotransporters in order to further investigate their translocation mechanism. Methods:Intimin from Escherichi coli was used as a model substrate. Spheroplasts were prepared from Escherichi coli strains overexpressing Intimin to induce the expression of Intimin. Recombinant β-barrel assembly machinery (BAM) complex was obtained and purified, and then proteoliposomes containing BAM were prepared. Following the digestion with proteinase K, the translocation was detected by SDS-PAGE. Results:Spheroplasts were induced to express Intimin, and then BAM-containing proteoliposomes were added to the system. Compared with control and liposomes groups, the experimental group showed that Intimin was resistant to proteinase K treatment, indicating that Intimin was successfully translocated.Conclusions:The translocation of Intimin required the participation of BAM complex. An in vitro reconstitution system for inverse autotransporters was constructed in this study, providing a method to study the translocation mechanism of inverse autotransporters.

Objective:To construct a surface display system containing various lengths of the Ag43 passenger domain for an optimal bacterial surface display of foreign protein HPV16L1.Methods:(1) Ag43 gene sequences of different lengths were inserted into pET22b vector to construct four Ag43 surface display vectors (Ag43/138, Ag43/551, Ag43/552 and Ag43/700) using PCR and subcloning strategy. (2) The generation of four HPV16L1-Ag43 fusion constructs was completed by PCR and subcloning methods. (3) HPV16L1-Ag43 fusion proteins were expressed and analyzed by SDS-PAGE. (4) The surface exposure of HPV-16L1 was verified using trypsin digestion.Results:PCR analysis and sequencing results showed that Ag43 surface display vectors and HPV16L1-Ag43 fusions were constructed successfully. SDS-PAGE showed that the expression of HPV16L1-Ag43 fusion proteins could be induced with 0.2 mmol/L IPTG and the protein content was reduced after the cells were treated with trypsin, especially the content of Ag43/700-HPV16L1 that showed a drastic reduction.Conclusions:The Ag43 surface display system was successfully constructed and could be used for a successful display of HPV16L1. This study also showed that Ag43/700 comprising only the α-helix and the β-barrel of Ag43 provided an optimal surface display for HPV16L1.

Objective:To screen active antibacterial components from licorice extract using BamA and BamD, the core components of Escherichia coli ( E. coli) β-barrel assembly machinery (BAM), as targets in order to combat the increasingly serious problem of antibiotic resistance. Methods:Affinity ultrafiltration combined with high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to screen the potential components interacting with BamA and BamD from licorice extract. Changes in the expression of bamA and bamD genes of E. coli after treatment with the compounds were detected by fluorescence quantitative PCR, and the effects of the compounds on the function of the BAM complex to integrate outer membrane proteins into the bacterial outer membrane were analyzed using an in vitro recombination system. The influence of the compounds on the integrity of bacterial membranes was evaluated through analyzing the accumulation of SDS within the bacterial cells. Results:Bioaffinity ultrafiltration combined with HPLC-MS screening revealed that 18β-glycyrrhetinic acid could interact with BamD. After 18β-glycyrrhetinic acid treatment, the expression of bamA gene increased by 1.5 times, and the expression of bamD gene increased by 2 times. However, the inhibitory effect of 18β-glycyrrhetinic acid on the membrane insertion function of the BAM complex was not observed in the in vitro recombinant system assay, and the cell membrane integrity assay experiments did not reveal any disruption of the E. coli cell membrane by 18β-glycyrrhetinic acid. Conclusions:Using BamA and BamD proteins as targets, a natural product screening method using affinity ultrafiltration combined with HPLC-MS is established. The screening result shows that 18β-glycyrrhetinic acid can interact with BamD and affect the expression of outer membrane proteins in E. coli. Therefore, the screening and experimental procedures established in this study are of good reference value for the screening of novel antimicrobial drugs from other sources targeting outer membrane proteins, and this study also suggests that the selection of the relevant target sites is crucial for the successful screening of the corresponding natural products.