Aim: To establish a prokaryotic expression system of the tandem repeat of CA125 (CA125R), express and purify the recombinant CA125R protein, prepare its antiserum.
Methods: The full gene sequence of one tandem repeat of CA125 was synthesized and cloned into pET-32a(+) to construct a prokaryotic expression vector of the CA125R protein (pET-CA125R). The pET-CA125R was transformed into E.coli BL21 (DE3) and the soluble expression conditions were optimized; the pure recombinant CA125R protein was prepared by affinity Ni-NTA chromatography and identified by Western blotting. A rabbit was immunized with the pure recombinant CA125R protein to prepare its antiserum.
Results: The prokaryotic expression vector of CA125R was successfully constructed. The optimal soluble induction expression conditions were 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) at 15DegreesCelsius for 6 h. Western blotting confirmed the pure CA125R recombinant protein of high purity. The prepared antiserum specifically recognized recombinant CA125R protein and natural CA125 glycoprotein.
Conclusion: We successfully established the efficient prokaryotic expression system of the CA125R, and prepared the recombinant CA125R protein of high purity and its antiserum.