Abstract: 【Objective】 This study was aimed to obtain specific nanobody against signal transducer and activator of transcription 3 (STAT3) protein and then validate its use for immunohistochemistry and Western blotting,as well as the function on the cell growth of B16 melanoma cell.【Method】 Anti-STAT3 nanobodies were screened from B16 nanobody library from alpaca by phage display.After the positive clones were obtained and sequenced,one clone was ligated into the prokaryotic expression vector pColdⅠto construct the plasmid which was expressed by E.coli and purified by nickel columns,molecular sieves and ultrafiltration tubes.Immunohistochemistry and Western blotting were used to verify whether anti-STAT3 nanobody bound STAT3 protein in brain tissue and had the effects on the proliferation of B16 cells by CCK8 method and related genes expression on the proliferation pathway by Western blotting.【Result】 After phage display screening and sequencing of positive clones,a monoclonal strain of nanobody specifically binding STAT3 protein was successfully obtained and named as STAT3-VHH-10B.The plasmid of the ligation of STAT3-VHH-10B and pColdⅠwas expressed and then purified,which was verified by SDS-PAGE and showed anti-STAT3 nanobody with the size of about 15 ku.The results of immunohistochemistry and Western blotting showed that the purified anti-STAT3 nanobody could detect STAT3 expression in brain tissue.The results of cell proliferation test showed that anti-STAT3 nanobody could significantly inhibit the proliferation of B16 cells,and down regulate the protein expression of the main genes on the cell proliferation pathway,including CycD1, Bcl2 and cMyc.【Conclusion】 A specific anti-STAT3 nanobody was successfully screened and could be used for immunohistochemistry and Western blotting to detect the expression of STAT3 in tissues.Moreover,it could inhibit the proliferation of B16 cells by inhibiting the expression of main genes CycD1, Bcl2 and cMyc on the basis of binding with STAT3.The results would provide a new tool for the functional research of STAT3 and its nanobody.

Key words: nanobody; signal transducer and activator of transcription 3 (STAT3); melanoma; B16 cells