Prokaryotic Expression, Polyclonal Antibody Preparation Of AZGP1and Application For Clinical Diagnosis In Hepatocellular Carcinoma

Objectives:Recent study found that AZGP1protein is closely related to hepatocellular carcinoma(HCC). To assess its value in the diagnosis of HCC and prepare monoclonal antibody, this study will recombinant express the AZGP1and prepare polyclonal antibody.Methods:The full length of AZGP1was amplified from HCC tissue and cloned into the prokaryotic expression vector pET21a(+)-MBP and then transformed into E.coli DH10B.The recombinant protein of AZGP1was expressed by the induction of isopropyl-β-d-thiogalactoside(IPTG) and purified by His-tag magnetic bead purification kit.Then it was detected by SDS-PAGE.Polyclonal antibody was developed by immunizing BALB/c mice with purified recombinant protein.The specificity and sensibility of antibody were determined by Western-blot and ELISA respectively. Moreover, the level of AZGP1protein in the serum of HCC patient was detected by Western-blot.Results:1.The full length of AZGP1was obtained from HCC tissue by RT-PCR.The prokaryotic expression plasmid pET21a(+)-MBP-AZGP1was successfully constructed and confirmed by PCR,endonuclease digestion and direct sequencing.2.By IPTG induction,recombinant protein MBP-AZGP1was expressed efficiently in the form of inclusion bodies in E.coli BL21.We obtained74kDa purified recombinant protein by His-tag magnetic bead purification kit. After renaturation, the recombinant protein MBP-AZGP1 had recovered its activity.3.We successfully obtained anti-AZGP1polyclonal antibody by immunizing BALB/c mice with purified recombinant protein.The highest titer of polyclonal antibody is1:12800by the detection of indirect ELISA.4.By Western-blot,we found that the level of AZGP1in the serum of HCC patients were significantly higher than healthy controls.Conclusion:1.The prokaryotic expression plasmid pET21a(+)-MBP-AZGP1was constructed. Recombinant protein MBP-AZGP1was successfully purified and renaturation.2.Anti-AZGP1polyclonal antibody with high specificity and sensitivity was obtained.3.The level of AZGP1was upregulated in the serum of HCC patients.