Aim: To further investigate the biological function of human novel gene CTRP4 by constructing the prokaryotic expression vector of human CTRP4, inducing the expression of and purifying hCTRP4-his protein in E.coli, and preparing polyclonal antibody against human CTRP4.
Methods: Human CTRP4 gene was amplified by PCR, digested with enzymes, and subcloned into a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-hCTRP4. The pET-32a-hCTRP4 was transformed into E.coli BL21(DE3). The hCTRP4-his fusion protein was induced by IPTG, purified by Ni-NTA purification system, and analyzed by SDS-PAGE. The recombinant vector pcDNA3.1-myc/his(-)B-hCTRP4 expressing full-length human CTRP4 and purified prokaryotic protein hCTRP4 were used to immunize BALB/c mice to produce polyclonal antibody. The anti-serum was purified and the characteristics of the antibody were identified by ELISA, Western blotting, immunofluorescence cytochemistry and immunohistochemistry.
Results: The prokaryotic expression vector of pET-32a-hCTRP4 was constructed successfully. hCTRP4-his fusion protein was expressed in E.coli BL21(DE3) after IPTG induction. The titer of the anti-serum reached 1:20 000, and its specificity was proved by Western blotting. The results of immunofluorescence cytochemistry and immunohistochemistry indicated that CTRP4 was mainly localized in the cytoplasm of hepatic cells.
Conclusion: hCTRP4-his fusion protein can be successfully expressed in E.coli. A specific polyclonal antibody against human CTRP4 has been successfully prepared.