丝裂原活化蛋白激酶15纳米抗体的制备及其在B16-F10黑素瘤细胞生长过程中的抑制作用  

Preparation of MAPK15 Nanobody and Its Inhibitory Effect on the Growth of B16-F10 Melanoma Cells

作　　者：贾琼 金聪俐 胡世雄 秦蓉芬 赵立峰 范瑞文[1] JIA Qiong;JIN Cong-Li;HU Shi-Xiong;QIN Rong-Fen;ZHAO Li-Feng;FAN Rui-Wen(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China;Beichen District Agricultural Development Service Center,Tianjin 300400,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030801 [2]天津市北辰区农业发展服务中心,天津300400

出　　处：《中国生物化学与分子生物学报》2024年第1期72-80,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：2021山西省研究生创新项目(No.2021Y318)资助。

摘　　要：丝裂原活化蛋白激酶15(mitogen-activated protein kinase 15,MAPK15),又称ERK7或ERK8,是MAPK家族的非典型新成员。MAPK15不同程度地促进不同肿瘤细胞的增殖、迁移、自噬等细胞活动。本研究以MAPK15为靶点,筛选特异性的MAPK15纳米抗体,评估其是否能够作为免疫组织化学和Western印迹中的一抗用于其抗原表达的检测,并探究该纳米抗体在B16-F10黑色素瘤细胞中的作用。通过噬菌体展示技术从B16-F10黑色素瘤细胞纳米抗体文库中进行筛选,得到1株MAPK15特异性纳米抗体,命名为MAPK15-VHH;将该菌株构建原核表达载体,进行优化诱导表达条件时发现,0.6 nmol/L IPTG,15℃,100 r/min条件下该纳米抗体的上清表达量最高。通过竞争ELISA法检测MAPK15-VHH的亲和力,结果显示,该抗体KD值为0.9829。通过Western印迹和免疫组织化学检测脑组织中MAPK15在蛋白质水平的表达量及分布情况,结果表明,MAPK15-VHH可与组织中的MAPK15结合,用于检测MAPK15蛋白的表达情况。在B16-F10黑色素瘤细胞中过表达MAPK15后向其中添加MAPK15-VHH,通过CCK8、Western印迹以及实时荧光定量PCR检测其对该细胞增殖和自噬的影响,结果显示,该MAPK15-VHH能作为MAPK15的拮抗剂,抑制B16-F10细胞的增殖和自噬,从而抑制黑素瘤细胞的生长。综上所述,本研究成功得到一株亲和力较强的MAPK15纳米抗体,可用于Western印迹及免疫组织化学以检测MAPK15在蛋白质水平的表达及分布;此外,该MAPK15纳米抗体可以作为MAPK15拮抗剂,有效地抑制B16-F10细胞的增殖和自噬进程,为临床检测试剂和治疗药物的开发提供理论基础。Mitogen-activated protein kinase 15(MAPK15),also known as ERK7 or ERK8,is an atypical 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 specific MAPK15 nanobody,then evaluated whether it could be used as primary antibodies in Western blotting 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 K D 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 overexpressing 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 distribution of MAPK15.In addition,the MAPK15 nanobody could serve as a MAPK15 antagonist to efficiently inhibit the proliferation and autophagy of B16-F10 cells,which provided a the

关 键 词：丝裂原活化蛋白激酶15 纳米抗体 黑色素瘤细胞 增殖 自噬 

分 类 号：Q7[生物学—分子生物学] S852[农业科学—基础兽医学]