Abstract:

Chitinase 3-like protein 1 (CHI3L1) expression levels significantly change after infection with influenza A virus (IAV), suggesting that it may play a role in regulating the antiviral immune response. However, no direct interaction between CHI3L1 and IAV has been found so far. Since CHI3L1 may play an important role in the immune process, researchers hypothesized that it may play a key role in regulating the antiviral immune response. However, the current research tools are insufficient. Therefore, there is an urgent need to develop an efficient method for detecting CHI3L1 to facilitate subsequent research. In this study, CHI3L1 was targeted as the protein of interest, and a eukaryotic expression plasmid was constructed for its successful in vitro expression and purification. The purified protein was immunized with alpacas. After four immunizations, peripheral blood was collected from the alpacas and lymphocytes were isolated, and the antibody level was determined to be 1:64 000, which met the standard for constructing phage display libraries. Then, a single heavy chain variable region (VHH) gene was amplified using nested PCR and cloned into the pComb phage vector, successfully constructing a library with a capacity of 1.2×10⁸ cfu·mL^-1 of CHI3L1 nanobody phage display library. Through three rounds of immune affinity selection and enrichment, four specific nanobodies (Nb) were selected. Subsequently, the CHI3L1-specific nanobody expression plasmid was further constructed and transfected into 293F cells for expression, and the protein was purified using the Protein A affinity purification system. The Nb-YC8 and Nb-YC4 showed good binding affinity as determined by ELISA. Western blot was used to further verify that these nanobodies were able to correctly recognize CHI3L1 protein compared to commercial antibodies. Immunofluorescence analysis showed that Nb-YC4, Nb-YD10 and Nb-YF8 could recognize CHI3L1 protein expressed in 293T cells, while Nb-YC8 failed to recognize CHI3L1, suggesting that it may recognize a linear epitope. In this study, four strains of CHI3L1-specific nanobodies with different affinities were successfully screened and identified, which are expected to be powerful tools to study the function of CHI3L1 in the body's immune system. Further studies of CHI3L1 may help to uncover its potential role in the antiviral immune response and provide a basis for the development of CHI3L1-based diagnostic or therapeutic approaches.

Key words: CHI3L1, nanobodies, characterization