人神经元特异性烯醇化酶基因克隆及单克隆抗体的制备与鉴定

Study on the molecular cloning of human neuron-specific enolase gene and preparation and identification of anti-NSE monoclonal antibodies

目的研究建立克隆人神经元特异性烯醇化酶(NSE)基因制备鼠抗人NSE单克隆抗体方法。方法用NSE特异引物,通过逆转录多聚酶链式反应从人肺癌细胞株A549中扩增NSE基因,构建重组质粒pGEMTNSE,并测序鉴定NSE基因序列。再将NSE基因定向克隆于原核高效表达载体pMS31b,当表达MS2NSE融合蛋白后,免疫BALB/C小鼠。并用常规杂交瘤技术,制备鼠抗人NSE单克隆抗体(NSEMcAb)。最后,采用免疫细胞化学(ICC)技术检测NSE在肿瘤细胞株中的表达。结果克隆岀全长1305bp的NSE基因,其表达的MS2NSE融合蛋白为57000,表达量142mg/L。获得2株能稳定分泌抗NSE单克隆抗体的杂交瘤细胞株,经检测其分泌抗体的亚型分别为IgG1和IgG2a。ICC检测证明所得NSEMcAb能检测到肿瘤细胞中NSE的表达。结论成功克隆了NSE基因,并获得鼠抗人NSEMcAb,为深入研究NSE基因的表达提供了有力的工具。

Objective To clone human neuron-specific enolase (NSE)gene and prepare the monoclonal antibodies against human neuron-specific enolase and to test the expression of NSE in tumor cell lines by immunocytochemistry (ICC).Methods The gene fragment of human NSE was amplified by RT-PCR and ligated to the pGEM vector. After the sequencing of recombinant NSE, it was ligated to the expression vector pMS-31b. The MS2-NSE fusion protein was expressed after higher temperature induction. The purified target protein was used for immunizing BALB/C mice to prepare McAbs against NSE.Results Full length of NSE gene with 1 305 bp was cloned. Molecular weight of MS2-NSE was 57 000. 1.42 mg/L of MS2-NSE fusion protein could be expressed. Two strains of hybridoma secreting NSE McAbs were obtained by ELISA screening. The subtypes of the NSE McAbs were IgG1and IgG2a. The two McAbs could react with A549 cell lines in ICC. NSE positive staining in ICC was mainly located in cytoplasm.Conclusions We clone human neuron-specific enolase gene, obtain the anti-NSE monoclonal antibodies and examine the expression of NSE in lung cancer tumor cell line.