Constructing a High-Diversity Nanobody DNA Library involves a two-phase blood collection process. The first round of blood collection occurs after the completion of the third antigen immunization, and the second round follows the fourth antigen immunization, with a total blood volume exceeding 200mL. Lymphocytes are isolated, and RNA extraction, along with the construction of the library, is performed.

Bacteriophage Surface Display Screening: Take an appropriate amount of constructed nanobody library, add bacteriophages after suitable cultivation. Nanobodies are displayed on the surface of bacteriophages in the form of fusion proteins with the pIII protein. After incubation with the antigen, washing, and elution, specific nanobodies are preliminarily obtained

After obtaining the positive nanobody sequence, the sequence is constructed into an expression vector for expression and purification. Due to the relatively simple structure of nanobodies, they can be correctly expressed and folded in prokaryotic cells. The expression and purification are performed using Escherichia coli BL21.

       Nanobodies can be utilized in real-time monitoring of receptor activity using the BRET method. According to the information documented in related articles, by constructing corresponding functional plasmids and conducting tests, the obtained results align with those presented in the articles. Leveraging BRET technology, nanobodies enable the real-time assessment of receptor activity.