[Preparation, characterization and application of polyclonal antibody against human carboxylesterases-II].

ABSTRACT

AIM: To construct a prokaryotic plasmid expressing human carboxyles-terases-II (hCE-II ), purify the recombinant protein and investigate the rabbit polyclonal antibody against hCE-II . METHODS: A recombinant plasmid expressing pGEX-4T-1-hCE-II (hCE-II -GST) and pET-32a- hCE-II (hCE-II -His) was constructed and then it was expressed in E.coli BL21 induced by IPTG. The polyclonal antibody was prepared by immunizing the rabbits with the purified recombinant protein. The sensitivity and specificity of the antibody was detected using enzyme -linked immunosorbent assay, immunohistochem ical staining and Western blot analysis. RESULTS: The polyclonal antibody against hCE-II was successfully prepared. Western blot analysis showed that the antibody specifically reacted with the recombinant protein and natural human liver microsomal proteins. Immunohistochemis result demonstrated that the pAb could combine with liver cytoplasm protein but not vascular smooth muscle cell protein. CONCLUSION: The successful preparation of the pAb against hCE-II with high titer and specificity lays a foundation for the investigation of diagnosis kit of liver cancer.