Method for preparing human neuron specific enolase (NSE) monoclonal antibody through gene immunization

Abstract

The invention discloses a method for preparing a human neuron specific enolase (NSE) monoclonal antibody through gene immunization. The method comprises the following steps of: performing RT-PCR (reverse transcription-polymerase chain reaction) with SEQ ID NO.1 and SEQ ID NO.2 as primers, thus obtaining SEQ ID NO.3 of an NSE gene; inoculating the NSE gene into pGEM-T to form plasmid pGEM-T-NSE; transfecting the plasmid to BEL7402 cells, cultivating the cells, purifying cell lysis buffer to obtain NSE recombinant protein; injecting mice with the plasmid; fusing splenocyte and myeloma cell of the immunized mice to obtain hybridoma cells; cultivating the hybridoma cells; screening positive hybridoma cells capable of generating a monoclonal antibody aiming at NSE protein epitope; injecting positive hybridoma cell strains into enterocoelia of the mice to generate ascites; purifying the ascites to obtain the human NSE monoclonal antibody. The method provided by the invention is capable of shortening the early screening time of the hybridoma cells and simplifying production, is simple to implement and needs a shorter period.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and molecular immunology, in particular to a method for preparing human neuron-specific enolase monoclonal antibody by gene immunization. technical background [0002] Human neuron-specific enolase (neuron-specific enolase, NSE) is one of the enolases involved in the glycolysis pathway, which exists in nerve tissue and neuroendocrine tissue. NSE had the highest activity in brain tissue cells, intermediate activity levels in peripheral nerves and neurosecretory tissues, and the lowest values ​​were found in non-nervous tissues, serum and spinal fluid. It was found to have excessive expression of NSE in tumors related to neuroendocrine tissue origin, especially in SCLC (small cell lung cancer), resulting in a significant increase in serum NSE in patients with small cell lung cancer. [0003] Clinical significance of neuron-specific enolase: [0004] Neuron-specific enolase (NSE) is a valuable m...

AI Technical Summary

Problems solved by technology

[0008] At present, most NSE monoclonal antibodies are prepared by prokaryotic expression to prepare antibodies. The affinity of antibodies against natural proteins has been poor, so that the practical value of this antibody is very low. The amount of secretion is extremely low, which also increases the difficulty of purification of natural NSE protein

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