论文部分内容阅读
目的:采用电穿孔辅助DNA免疫方法制备小鼠抗人附睾蛋白4(HE4)单克隆抗体(mAb)并对其生物学特性进行鉴定。方法:利用逆转录多聚酶链反应(RT-PCR)从卵巢癌患者组织中获得人HE4基因编码序列,将其亚克隆至pPICZαA表达载体中并测序鉴定。采用活体电穿孔法免疫BALB/c小鼠。单抗制备采用B淋巴细胞杂交瘤技术进行细胞融合,经多次克隆化培养,筛选出特异分泌鼠抗人HE4mAb的杂交瘤细胞株。采用Westernblot,Ig亚型分析和mAb表位分析等对mAb的生物学特性进行鉴定。结果:获得2株持续、稳定分泌鼠抗人HE4mAb的杂交瘤细胞株,命名为1-7-C和3-12-C。ELISA和Westernblot结果均显示本实验获得的2株mAb能够特异识别有天然构象的HE4蛋白,2株mAb的Ig亚类均为IgM,轻链均为λ链。结论:成功地构建了pPICZαA-HE4表达载体,并获得2株鼠抗人HE4杂交瘤,其所分泌的抗体能特异地识别HE4蛋白。
OBJECTIVE: To prepare the mouse anti-human epididymal protein 4 (HE4) monoclonal antibody (mAb) by electroporation-assisted DNA immunization and to evaluate its biological characteristics. Methods: Human HE4 gene coding sequence was obtained from ovarian cancer tissues by reverse transcription polymerase chain reaction (RT-PCR), subcloned into pPICZαA expression vector and identified by sequencing. BALB / c mice were immunized by electroporation in vivo. Monoclonal Antibodies The B lymphocyte hybridoma technique was used for cell fusion. After multiple cloning and culture, the hybridoma cell line secreting mouse anti-human HE4 mAb was screened out. The biological characteristics of mAb were identified by Western blot, Ig subtype analysis and mAb epitope analysis. Results: Two hybridoma cell strains that consistently and stably secreted mouse anti-human HE4 mAb were obtained and named as 1-7-C and 3-12-C. The results of ELISA and Western blot showed that the two mAbs obtained in this experiment could specifically recognize HE4 protein with natural conformation. The Ig subclasses of the two mAbs were both IgM and the light chains were both λ chain. CONCLUSION: The pPICZαA-HE4 expression vector was successfully constructed and two murine anti-human HE4 hybridomas were obtained. The secreted antibodies could specifically recognize HE4 protein.