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.