Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.

Objective To summarize clinical experience of application of muliplefoliated flap combined with tendon graft from dorsal foot for repairing soft tissue defects of multi-fingers. Methods Based on the results of microdissection researches, muliplefoliated flap including medial foot flap, the first metatarsal dorsal flap, lateral foot flap, extensor pollicis brevis of toe and extensor proprius of the fifth toe pedicled by anterior tibial vessels and dorsal pedis vessels as trunk, anterior medial malleolus vessels, the first metatarsal vessels and anterior lateral malleolus vessels as branches respective was designed for repairing soft tissue and tendon defects of multi-fingers. Results In all 12 cases, total survival of flap was 11 cases , 1 case had partial necrosis of distal flap and it was healed by dress changing. Conclusion Transplantation of muliplefoliated flap combined with tendon graft from dorsal foot is a good method to repair soft tissue and tendon defects of multi-fingers.

AIM:To investigate the molecular mechanism of a novel bromobenzene substituted trifluoromethylbenzo Cyclopentanone WW02 inhib-iting the viability and proliferation of human lung cancer A549 and H1299 cells.METHODS:The ef-fect of different concentrations of WW02(6.25,12.5,25,50 μg/mL)on cell viability and prolifera-tion of A549 and H1299 were measured using CCK-8 and EdU methods.After 24 hours of stimulation of A549 and H1299 cells with different concentra-tions of WW02,the changes in Akt and mTOR phos-phorylation levels under different concentrations of WW02 were detected through Western blot as-say.Macromolecular docking was carried out be-tween WW02,AKT and mTOR through MOE Dock.RESULTS:After treating A549 and H1299 cells with WW02 using different concentrations(6.25,12.5,25,50 μg/mL),the activity of A549 and H1299 cells decreased in a concentration dependent manner compared with the DMSO control group(P<0.05).The proliferation of cells showed a concentration dependent decrease compared to the DMSO con-trol group(P<0.05).Compared with the DMSO con-trol group,after 24 hours of WW02 stimulation,the phosphorylation levels of Akt and mTOR in A549 cells decreased under the concentration of WW02(12.5,25,50 μg/mL,P<0.05).Compared with the DMSO control group,the phosphorylation levels of Akt and mTOR in H1299 cells decreased af-ter 24 hours of WW02 stimulation(25,50 μg/mL,P<0.05).Based on pattern analysis,it was found that WW02 had a strong binding with Akt and mTOR,with the highest score of-8.3 kcal/mol for WW02 and mTOR,while the highest score for WW02 and Akt was-7.3 kcal/mol.CONCLUSION:WW02 inhib-its the activity and proliferation of lung cancer A549 and H1299 cells,and its mechanism of action may be achieved by directly binding to Akt and mTOR proteins to inhibit Akt and mTOR phosphory-lation.