AUTHOR=Qiu Jianxiang , Li Jie , Zhang Zhen , Dong Shirui , Ling Xiaomei , Fang Zhixin , Ling Quanshou , Huang Zhixin 

TITLE=Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1207048

DOI=10.3389/fbioe.2023.1207048

ISSN=2296-4185

ABSTRACT=<p><bold>Introduction:</bold><italic>Drosophila melanogaster</italic> is a model organism for studying developmental biology and human neural disorders. Nanobodies are the variable domains of the heavy chains of camelid heavy-chain antibodies (VHHs) with high affinity to their antigens and have applications in basic research, similar to traditional antibodies. In addition, nanobodies acting as functionalized antibodies or protein binders have become an additional valuable approach in <italic>Drosophila</italic>. This study aimed to develop a VHH library against <italic>Drosophila</italic> proteins and confirm its availability by retrieving some <italic>Drosophila</italic> protein-specific nanobodies from the library.</p><p><bold>Methods:</bold> An alpaca was first immunized with <italic>Drosophila</italic> embryo lysate and then its lymphocytes were isolated. Total RNA was extracted and cDNA was synthesized. The vhh sequences were amplified by two round PCR, which were then ligated to a phage display vector pADL-10b. The ligation products were transduced into SS320 competent cells to generate a VHH library. From this library, nanobodies against CG7544, Myc, and CyclinE was enriched and screened by phage display technology and ELISA. DNA sequences of identified nanobodies were cloned into pADL-10b-Flag-His for expression and purification in <italic>Escherichia coli</italic> SS320. Binding ability of purified nanobodies with corresponding antigens were determined by ELISA and surface plasmon resonance <italic>in vitro</italic>.</p><p><bold>Results:</bold> In this study, an immune VHH library against <italic>Drosophila</italic> embryo proteins was generated with a capacity of 3 × 10<sup>7</sup>. From this library, eight nanobodies against three <italic>Drosophila</italic> proteins, Myc, CyclinE, and CG7544, were identified and the DNA sequences of these nanobodies were obtained. These nanobodies were successfully expressed and purified from <italic>Escherichia coli</italic> SS320, and were demonstrated to bind corresponding antigens with high affinity <italic>in vitro</italic>. Moreover, the equilibrium constant between the highest enriched nanobodies and corresponding antigens were calculated.</p><p><bold>Conclusion:</bold> In summary, we report the availability of an immune VHH library and a highly efficient panning strategy for nanobodies against proteins in <italic>Drosophila</italic>.</p>