Expression Of Human Recombinant Heparanase In E.coli And Production Of Monoclonal Antibodies Against Human Heparanase

Objective: Recent studies showed that heparanase (HPSE) plays an important role in tumormetastasis, but many details remain unkown. In order to study the role of HPSE intumorigenesis, invasion, metastasis, and angiogenesis, it is necessary to product HPSE andits antibody.Methods: Using the target nucloetide sequence released by GenBank, two primers weredesigned to clone the full-length fragment cDNA of HPSE. The full-length human HPSEgene was cloned into the expression vector, pET28a(+), the recombinant plasmid wastransformed into E.coli. After transformation, E.coli BL21(DE3，pET28a(+)-HPSE) wasinduced to express human HPSE protein by IPTG. Different induction time, the properconcentration of IPTG and different media which may influence the expression level ofhuman HPSE protein were discussed. HisTrap TMcrude column was used to purifyrecombinant HPSE protein.HPSE protein (50kDa) was used to immune BALB/c. After about one month, SP2/0and B lymphocyte were infused by hybridoma technique. Indirect ELISA was used todetect the secretion of antibody, the antigens for ELISA were full-length human HPSEprotein, HPSE protein and polypeptide Lys158-Cys172of HPSE (50kDa). The polypeptidehas the activity site of HPSE that binds to the heparin. HPSE positive clones wererepeatedly screened and subcloned. The specificity of monoclonal antibodies against humanHPSE was detected by Western blot. Results: Full-length recombinant human HPSE protein was expressed in E.coli. When therecombinant E.coli was incubated in2×YT media, the concentration of IPTG was1mmol/L, after5h inducing, the highest expression level of HPSE was obtained. Two celllines that secret monoclonal antibodies against human HPSE were cloned. The titers ofMcAb4D12、3F5were higher than1.28×105, their affinities were2.71×109M-1、6.67×108M-1，respectively.4D12had reactivity with full-length human HPSE protein，HPSE protein(50kDa).3F5had reactivity with full-length human HPSE protein，HPSE protein (50kDa)and a peptide with15amino acid residues Lys158-Cys172from N-terminus of HPSE(50kDa).Conculusion: The prokaryotic expression system for human HPSE was constructedsuccessfully. Two cell lines that secret monoclonal antibodies against human HPSE wereobtained.