Tang Mei-Ling, Xiao Ping, Zou Ji-Zhen, Cao Ding-Ding, Li Yuan-Yuan, Chang Hui-Bo
Department of Biochemistry and Immunology, Capital Institute of Pediatrics, Beijing 100020, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2018 Jun;20(6):501-507. doi: 10.7499/j.issn.1008-8830.2018.06.014.
To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.
A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone.
The LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05).
LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.
制备LINE1-ORF1p多克隆抗体，并研究LINE1-ORF1p对肾母细胞瘤WT_CLS1细胞增殖的影响。
采用基因工程方法实现LINE1-ORF1p的原核表达，并用LINE1-ORF1p免疫家兔制备多克隆抗体。采用间接ELISA法评估抗体效价，采用Western blot和免疫组织化学法评估抗体识别LINE1-ORF1p的特异性能力。构建真核表达载体pEGFP-N1-LINE1-ORF1并用于转染WT_CLS1细胞。分别采用Western blot和qRT-PCR法检测LINE1-ORF1的蛋白和mRNA表达，采用细胞增殖实验和集落形成实验评估LINE1-ORF1p对WT_CLS1细胞增殖及肿瘤细胞克隆形成的影响。
制备的LINE1-ORF1p抗体效价>1:16 000，能特异性识别细胞和肿瘤组织中的LINE1-ORF1p。转染pEGFP-N1-LINE1-ORF1的WT_CLS1细胞中LINE1-ORF1的mRNA和蛋白表达显著增加，细胞增殖能力和集落形成能力显著增强（P<0.05）。
LINE1-ORF1p可促进肾母细胞瘤细胞生长及肿瘤细胞克隆形成，可能参与肾母细胞瘤的发病机制。
