Study on the molecular cloning of human neuron-specific enolase gene and preparation and identification of anti-NSE monoclonal antibodies / 中华检验医学杂志

ABSTRACT

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.