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. 2016 Dec 26;7:649. doi: 10.3389/fimmu.2016.00649

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Copyright © 2016 Chu, Zhu, You, Zhang, Zheng, Cai, Zhou, Wang, Sun, Yang, Zhang, Wang, Nie, Zhu and Wang.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Preparation of the hS9-Fab03. (A) The heavy chain PCR products of positive clone. Lane 1, V_H; Lane 2, C_H; Lane 3, V_H combined with C_H; Lane M, DNA maker. (B) The light chain PCR products of positive clone. Lane 1, V_L; Lane 2, C_L; Lane 3, V_L combined with C_L; Lane M, DNA maker. (C) The construction of pETDuet-hSiglec-9 Fab03. Lane 1, the plasmid without restriction endonuclease digestion; Lane 2, the plasmid was double digested with NcoI and XhoI; Lane 3, NcoI and HindIII were used for double digesting the plasmid; Lane 4, EcoRV and XhoI were used for double digesting the plasmid; Lane M, DNA marker. Coomassie blue staining (D) and Western blotting (E) detected the expression of the recombinant vector. Lane 1, whole lysate of untransfected E. coli BL21, as a negative control; Lane 2, whole lysate of pETDuet-hSiglec-9 Fab03-transfected E. coli; Lane 3, supernatant of sonicated lysate of pETDuet-hSiglec-9 Fab03-transfected E. coli; Lane 4, sediment of sonicated lysate of pETDuet-hSiglec-9 Fab03-transfected E. coli; Lane M, protein marker. All strains were induced by IPTG overnight. Coomassie blue staining (F) and Western blotting (G) detected the purified Fab fragments. Lane 1, supernatant of sonicated lysate of pETDuet-hSiglec-9 Fab03-transfected E. coli induced by IPTG overnight; lane M, protein marker. (H) Coomassie blue staining showed that the hS9-Fab03 was expressed, and the heavy chain Fd and light chain L were linked together. (I) horseradish-peroxidase-conjugated goat anti-human antibody (Fab specific) was used to detect the heavy chain Fd, and light chain L of the hS9-Fab03 was expressed and separated in Western blotting. Coomassie blue staining (J) and Western blotting (K) detected the efficiency of hS9-Fab03. Lane 1, supernatant of sonicated lysate of the pETDuet-hSiglec-9 Fab03-transfected E. coli was purified by the His-trap Lambda Fab Select column; lane 2, whole lysate of pETDuet-hSiglec-9 Fab03-transfected E. coli induced by IPTG overnight; lane M, protein marker.