Screening For Nanobodies Targeting SIX1

Objective: SIX1 is expressed in various tumors,and its expression is positively correlated with tumor grades.Nanobodies have the advantages of low immunogenicity,low molecular weight,ease of preparation,and high stability.In this study,we screened the yeast surface-displayed nanobody library for nanobodies targeting SIX1,and preliminarily determined the in vitro activity of a SIX1-targeting dodecapeptide.Methods: Firstly,prokaryotic expression and purification of SIX1 protein were carried out,and used as an antigen for screening: SIX1 protein was labeled with either FITC or PE,incubated with the yeast-displayed nanobody library,and magnetic beads coated with FITC/PE antibody were added and sorting was carried out in an automatic immunomagnetic bead cell sorter.FITC and PE were screened for two rounds respectively,for a total of four rounds.The positive cells were collected and the positive rate was analyzed by flow cytometry.The positive clones obtained were further enriched by fluorescence-aided cell sorting.To monitor the false positive rate,a negative control group in which yeast was not induced to express nanobodies was set up for the whole screening process in parallel,and the monoclonal extraction plasmids were obtained from this group and subjected to sequencing.Moreover,also targeting SIX1,the effect of a dodecapeptide PDP2 on the proliferation of mouse pancreatic cancer cell line pan-02 was detected by the CCK method.Results: The positive rate of FITC magnetic bead sorting was 13.38% in 2 rounds,and the positive rate of PE in the following 2 rounds increased to 34.6%,indicating that magnetic bead sorting effectively enriched positive clones.Although the second round of FITC screening had not significantly changed the positive rate,the second round of PE(34.6%)significantly increased the positive rate compared with the first round(7.19%),indicating that the second round of enrichment was necessary.After flow sorting,a total of 1440double-positive clones were collected.Clones were also obtained from the negative control group,indicating the existence of false positives.In addition,the dodecapeptide PDP2 was found to significantly inhibit cell proliferation at 50,100,and 150 μM.Conclusion: The combination of magnetic beads and fluorescence-aided cell sorting can successfully yield multiple clones targeting SIX1.The monitoring of the positive rate indicates that each label should be screened for at least two rounds.The negative control group indicates that there are false positives,and the obtained monoclonal should be further characterized.The dodecapeptide PDP2 targeting SIX1 can significantly inhibit the proliferation of mouse pancreatic cancer cells.