葡萄胎病理相关基因F10编码蛋白单克隆抗体的制备与应用

Preparation and application of monoclonal antibody of a new gene F10 associated with the pathogenesis of hydatidiform mole

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。

To express the recombinant protein for a new hydatidiform mole-related gene F10 and to prepare the F10 monoclonal antibody（m Ab） by immunizing mice,the coding sequence of F10 gene was amplified by reverse transcription polymerase chain reaction（RT-PCR） from human tissue and cloned into the vector p ET28 a for constructing a prokaryotic plasmid. Then the plasmid was expressed in the E. coli BL21（DE3） to obtain the F10 recombinant protein,which was subsequently purified and injected to Balb/c mice to produce anti-F10 m Ab.Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-F10 m Ab were subcloned with limited dilution. Enzyme-linked immunosorbent assay（ELISA） was applied to evaluate the titers and subtypes of the m Ab. Immunohistochemistry was applied to detect the expression of F10 in tissues of hydatidiform mole and normal placenta to check the specificity of F10 m Ab. The data showed that the prokaryotic plasmid expressing F10 recombinant protein was successfully constructed; and three hybridoma cell lines secreting anti-F10 m Ab were obtained. The titer of the F10 m Ab in ascites was 1 ∶7.2 ×10^5,and the subtype of the F10 m Ab was Ig G1. Immunohistochemistry analysis using the F10 m Ab prepared showed the expression level of F10 in hydatidiform mole was significantly higher than that in normal placenta. In conclusion,the F10 recombinant protein with strong antigenicity is expressed and highly purified,while the anti-F10 m Ab is also prepared successfully,which possesses high titre and specificity.