pMES4

Superfolder green fluorescent protein (sfGFP) is a fusion tag that plays a dual role in monitoring and purifying recombinant fusion proteins using specific conjugates. Nanobodies are the smallest intact antigen-binding fragments of pure heavy chain antibodies (HCAbs) derived from camelid species. They are produced as recombinant proteins in E. coli and have different biotechnological applications, including detection and purification of their specific antigens. To generate anti-sfGFP-specific nanobodies, the researchers successfully immunized an adult dromedary camel and evaluated the immune response by ELISA, which showed that HCAbs are actively involved in the response.

To construct a nanobody "immune" library, large blood samples were collected from immune camels, and lymphocytes were isolated to extract RNA. Total RNA was converted into cDNA by reverse transcription reaction. Two subsequent PCR reactions were performed on this cDNA template to amplify the nanobody genes. The purified Nanobody gene and pMES4 phagemid were digested with PstI and BstEII restriction enzymes and then ligated using T4 DNA ligase. The ligation product was transformed into newly prepared electrocompetent E. coli TG1 cells, cultured at 37°C overnight, and the growing colonies were scraped. A relatively large Nanobody "immune" library was finally prepared, containing 5×10⁸ individual transformants, with a positivity rate of 87.5%.

Figure 1. Constructing of anti-sfGFP nanobodies library and panning by phage display. (Twair, Aya, et al., 2014)