Cloning of asparagine synthetase gene and preparation,identification of the monoclonal antibody against asparagine synthetase / 中华检验医学杂志

ABSTRACT

Objective To clone human asparagine synthetase(ASNS)gene,express the MS2- ASNS fusion protein through gene engineering and use the purified target protein to immune BALB/C mice, prepare and identify the monoclonal antibody(McAb),which forms the base for studying mechanism of L- asparaginase used as salvage regimen in midline NK/T cell lymphoma nasal type.Methods ASNS gene fragment was amplified by RT-PCR from HepG2 cell line and constructed into prokaryocytic expression vector.Fusion-protein of MS2-ASNS was expressed and used for immunizing BALB/C mice to prepare McAbs against ASNS.Identified the McAb and detected the expression of ASNS in tumor.Results Part of the ASNS reading frame(NCBI,M27396179-1 420 bp)was cloned and product length of RT-PCR was 1 263 bp.Molecular weight of MS2-ASNS was about 54 700 Da.Two strains of hybridoma secreting ASNS McAbs were obtained.The subtype of the ASNS McAb was IgG2a.Western-blot showed that the McAbs could specifically react with MS2-ASNS fusion protein and ASNS protein in tumor cell lines.ASNS expression was detected by immunocytochemistry and Immunohistochemisrry.Conclusion We have cloned human ASNS gene,obtained the anti-ASNS McAb and examined the expression of ASNS in tumor.