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目的构建原核质粒表达葡萄胎相关新基因F10重组蛋白,分离纯化制备其单克隆抗体,并鉴定其生物学特性。方法用逆转录聚合酶链反应法从成人组织中扩增F10编码序列,将其克隆到p ET28a原核表达载体,在大肠杆菌BL21(DE3)内进行诱导表达F10基因重组蛋白。以纯化后的重组蛋白免疫Balb/c小鼠制备单克隆抗体。ELISA法检测杂交瘤细胞分泌F10单克隆抗体的效价和类型,免疫组织化学染色检测F10蛋白在葡萄胎及胎盘组织中的表达,鉴定制备F10单克隆抗体的特异性。结果成功构建了p ET28a/F10原核表达质粒并获得高纯度的重组F10蛋白。筛选出3株能稳定分泌抗F10蛋白的单克隆抗体杂交瘤细胞系,其效价比高达1∶7.2×105,免疫球蛋白类型均为Ig G1类。免疫组织化学染色显示该抗体能特异结合葡萄胎及胎盘绒毛组织中的F10蛋白,且葡萄胎组织中F10蛋白的表达显著高于正常胎盘组织。结论表达及纯化的重组F10蛋白纯度高,免疫原性强。以此为抗原制备的抗F10蛋白的单克隆抗体效价高、特异性强,可用于F10功能的进一步研究。
Objective To construct prokaryotic plasmids expressing novel F10 recombinant protein of hydatidiform mole, isolate and purify its monoclonal antibody and identify its biological characteristics. Methods The F10 coding sequence was amplified from adult tissues by reverse transcription polymerase chain reaction and cloned into prokaryotic expression vector p ET28a. The recombinant F10 protein was induced in E. coli BL21 (DE3). Monoclonal antibodies were prepared by immunizing Balb / c mice with the purified recombinant protein. The titer and type of F10 monoclonal antibody secreted by hybridoma cells were detected by ELISA. The expression of F10 protein in hydatidiform mole and placenta was detected by immunohistochemical staining and the specificity of F10 monoclonal antibody was identified. Results The p ET28a / F10 prokaryotic expression plasmid was successfully constructed and a high purity recombinant F10 protein was obtained. Three monoclonal antibody hybridoma cell lines that can stably secrete anti-F10 protein were screened out. The titer of the hybridoma cell line is 1: 7.2 × 105, and the immunoglobulin types are all Ig-G1. Immunohistochemical staining showed that the antibody could specifically bind F10 protein in hydatidiform mole and placental villi, and the expression of F10 protein in hydatidiform mole was significantly higher than that of normal placenta. Conclusion The recombinant F10 protein expressed and purified has high purity and strong immunogenicity. The anti-F10 protein monoclonal antibody prepared by the antigen has high titer and strong specificity and can be used for further research on the function of F10.