Neuron-specific Enolase Gene Cloning And Monoclonal Antibody Preparation And Identification

Background Neuron-specific enolase is one isoenzyme of enolase. Isoenzymes which are composed of γγ and αγ specially exist in neur and neuroendocrine cells, so they were named as Neuron-specific enolase. The cDNA, was composed of 2423 bp of nucleotides, 1305 bp of an open reading frame encoding a protein of 434 amino acids .Objects This paper is to clone the human neuron-specific enolase gene, prepare McAbs against NSE and to identify them and to examine the expression of NSE in tumor cell lines by immunohistochemistry (IHC). Method First, using NSE specific primer and by RT-PCR, the full length of cDNA of NSE moleculer was obtained through human lung cancer cell strain A549, then recombined plasmid pGEM-T-NSE was constructed and was sequenced to identify the gene sequence. Second, NSE gene was ligated to the high effective expression vector pMS-31b, then pMS-31b-NSE was transformed into colibacillus POP2136 and after higher temperature induction, the fusion protein MS2-NSE was expressed. After being separated by SDS-PAGE electrophoresis, being eluted and being purified through dialysis, the fusion protein was used to immunize Balb/c mice and two strains of hybridoma secreting McAbs to NSE were obtained through routine hybridoma technology and ELISA screening, then was injected into abdominal cavity of mice and was cultured to prepare ascites. The subtypes of secreting NSE McAbs against NSE were examined and different methods of purification were selected according to different subtypes. Third, the prepared McAbs were identified by cytochemistry, specificity and Western-blot. The prepared McAbs were used to test the expression of NSE in human lung cancer cell strain A549, BE1(high transfered), LH7(low transfered) and in liver cancer cell strain 7402 by IHC technology. Results the human neuron-specific enolase gene was cloned and the result of sequence examination proved the gene sequence of NSE gene which was cloned was completely correct, two strains of hybridoma secreting McAbs to NSE stably were obtained, anti-NSE Monoclonal Antibodies were obtained whose subtypes were IgG1 and IgG2a respectively. NSE was expressed in all of human lung cancer cell strain A549, BE1(high metastasis), LH7(low metastasis) and liver cancer cell strain 7402. Conclusion we obtained the anti-NSE monoclonal antibodies and examined the expression of NSE in tumor cell lines,which established a basis for the following research.