Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

Screening scFv Specific to Vcam-1 by Phage Display Library and Its Activity Evaluation / 华中科技大学学报(医学版)

Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones were transferred into E.coli ,and the clone with the highest expression was regarded as the final requisite one.Competent cells were infected by the final requisite clone and scFv was expressed.After purification ,the activity of scFv was tested by ELISA and its affinity was evaluated.Results Molecular weight of Vcam‐1 antigen protein was 85-90 kD.Positive clones were screened out by taking Vcam‐1 protein as the antigen ,and 9 high titer clones were obtained by single phage ELISA.Gene sequencing of these clones was carried out and 3 sequences were obtained ,1 of which got the highest expression.Molecular weight of the expressed scFv was about 30 kD.The scFv got high affinity to Vcam‐1 antigen according to ELISA ,in spite of its lower activity than full‐length monoclonal antibody.Conclusion scFv antibody specific to Vcam‐1 was successfully obtained from phage display librar‐y ,which laid the foundation of subsequent in vivo diagnosis and therapy.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

Strategy Analysis of Antibody Industrialization in China / 中国生物工程杂志

Recombinant antibodies have become the major growth trends in biotech industry following their success on therapeutic application and good revenue. But the low level of mammalian expression and laggard fermentation process constrained the development of antibody industry in China. The global advances of antibody industry were reviewed, compared the respective advantage between dihydrofolate reductase and glutamine synthetase expression system, continuous perfusion and fed-batch processes were compared. Finally, based on the knowledge and experience of antibody expression and fermentation, the suitable strategy of antibody industrialization, e.g. the fermentation model and scale, should depend on the comprehensive consideration of entrepreneur for the productivity, manufacturing capacity and market revenue. It may be a wise choice to use glutamine synthetase expression system and continuous perfusion process for the need of Chinese antibody industrialization.

The recombinant antibody HBsAg-Fab of hepatitis B virus to block hepatitis B reinfection after liver transplantation: a research in vitro / 中国普通外科杂志

Objective To study the effect of recombinant antibody HBsAg-Fab of hepatitis B virus(HBV) to block hepatitis B reinfection after liver transplantation.Methods The functional efficiency of antibody Fab in blocking hepatitis B reinfection after LT was analysis and studied by vitro infection test,complement toxicity assay and virus infection test,calculated the antibody absorption rate and cell death rate and cell infection rate,Results When the group with and the group without recombinant antibody Fab were compared,the antibody absorption rate,cell death rate and cell infection rate between the 2 groups showed sigificant defference(P

Study on the construction and expression of anti-IL2R? genetically engineering an-tibody Fab / 中国免疫学杂志

Abstract Objective: To construct and express anti-IL-2R? genetically engineering antibody Fab and identify its biological activity.Methods: PCR were used to construct cloning vector 5G1-pA22 of anti-IL-2R? antibody Fab,by digestion of vector 5G1-pA22, anti-IL-2R? an-tibody Fab gene fragment was obtained and then this fragment was coupled with digesting product of pET28b plasmid by EcoRI and HindIII toget a recombinant expression vector 5G1-pET28b, it was highly expressed in the E. coli BL21(DE3) in the form of inclusion bodies, compoing20% of the total protein. After the renaturation of antibody Fab, its binding activity was identified by ELISA and antibody competitive inhibitionassay in vitro. Results: Anti-IL-2R? antibody Fab was successfully constructed and highly expressed in the form of inclusion bodies, its yield wasup to 1 mg/ml.When applied to ELISA, antibody Fab exhibited good spectfic reactivity with rIL-2R, antibody competitive inhibition assay invitro indicated that antibody Fab can compete with IL-2 for binding IL-2R in activated T cell surface.As a result,the activation and proliferationof T cells were inhibited and inhibition percentage exceeded 90%. Conclusion: Had successfully constructed and expressed anti-IL-2R? geneti-cally engineering antibody. Fab,it had good biological ativity.