Selection Of Inhibitory Nanobodies Against Alliinase From Bactrian Camel Vhh Library

The development of new agents target to enzyme is alluring,and with the recent advances in molecular biology, there comes the needs to develop more agents based on specific inhibitors. In several species of the Camelidae family, a unique type of heavy chain antibody (HCAb) naturally devoid of L-chains was discovered. Although these peculiar antibodies lack light chains and also the CH1-domain, the CDR3 region of these antibodies possesses the extraordinary capacity to form long loops that can penetrate into the active site of enzyme. Research revealed that VHH (variable domain of the heavy chain of HCAb) single domain antibodies from phage display antibody library acquired the potential to recognize protein cavities and as such the ability to inhibit enzymes. In addition, VHHs can be easily cloned from camel and have been successfully produced as recombinant proteins, and usually called single domain antibodies (sdAbs). When compared with the conventional tetrameric IgGs, VHH bears several unique features, such as: lower molecular weight, more highly soluble and stable, lower antigenicity and better tissue penetration. These properties make VHH antibodies an alternative candidate as an enzyme inhibitor in therapeutic application.In our country, camelus bactrianu are mainly distributed in the western areas such as XinJiang, QingHai, and Inner Mongolia. Till now, it hasn’t been seen yet that a report about VHH from Camelus bactrianu. So in this respect, we try to study the functional VHH from this kind of camel in this research. Firstly, we cloned the VHH fragments of two HCAb subtypes from camel immunized with alliinase and ligated to phagemid pCANTAB 5E. The fragments are about 400 bp~600 bp. Then the recombinat phagemids were transformed into E.coli TG1 and rescued with M13KO7 phages. Therefore a phage display VHH library of 2.8×105cfu was constructed. With three cycles of biopanning, one VHH fragment specific to alliinase designated VHH A4 was selected and expressed. The specific binding of GST-VHH A4 to alliinase was found by Western blot analysis. Furthermore, VHH A4 antibody has been shown to markedly inhibit the enzymatic activity of alliinase at a low concentration. The results indicate that VHH library might be an optimal platform for screening potent enzyme inhibitors. Since the production of allicin with antitumor activity was catalyzed by alliinase and the inhibitors of alliinase play a great role in preparation of abzyme, which might be an alternative strategy in site-directed cancer treatment.