Preparation of MAPK15 Nanobody and Its Inhibitory Effect on the Growth of B16-F10 Melanoma Cells
Mitogen-activated protein kinase 15(MAPK15),also known as ERK7 or ERK8,is an atypi-cal new member of the MAPK family.MAPK15 promotes cancer cell proliferation,migration,autophagy and other cellular activities in different tumors at different degrees.In this research,we screened for spe-cific MAPK15 nanobody,then evaluated whether it could be used as primary antibodies in Western blot-ting and immunohistochemistry for the detection of its antigen expression,and investigated its role in B16-F10 melanoma cells.The results showed that a strain of MAPK15-specific nanobody was screened from B16-F10 melanoma cell nanobody library through phage display technology,which we named MAPK15-VHH.The strain was constructed into a prokaryotic expression vector,and we found that the nanobody expression level in the supernatant was the highest under the conditions of 0.6 nmol/L IPTG,15℃,and 100 r/min basing on optimizing the induction expression conditions.The affinity of MAPK15-VHH was detected by competitive ELISA,and the results showed that the KD value of MAPK15-VHH was 0.9829.MAPK15-VHH was used as the primary antibody in Western blotting and immunohistochemistry assays to detect the expression and distribution of MAPK15 in brain tissues by binding to MAPK15.After overex-pressing MAPK15 in B16-F10 melanoma cells,MAPK15-VHH was added to the medium,and its effect on cell proliferation and autophagy was detected by CCK8,Western blotting and quantitative real-time PCR.The results showed that the MAPK15-VHH could act as an antagonist of MAPK15 to inhibit the proliferation and autophagy of B16-F10 cells,thereby inhibiting the growth of B16-F10 melanoma cells.In summary,the MAPK15 nanobody obtained in this research had strong affinity,which could be used as a primary antibody in Western blotting and immunohistochemistry assays to detect the expression and dis-tribution of MAPK15.In addition,the MAPK15 nanobody could serve as a MAPK15 antagonist to effi-ciently inhibit the proliferation and autophagy of B16-F10 cells,which provided a theoretical basis for the development of clinical detection reagents and therapeutic drugs.
mitogen-activated protein kinase 15(MAPK15)nanobodymelanoma cellproliferationautophagy
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