ABSTRACT
Objective To express gastric cancer related gene GCRG213 by using thioredoxin fusion expression system, and to prepare human GCRG213 fusion protein. Methods GCRG213 cDNA with complete open reading frame was amplified by PCR from plasmid pGEM-T, and then was cloned into thioredoxin fusion expression vector pET102/D-TOPO. The recombinant plasmid was further transformed into E.coli BL21 strain. After induction with IPTG, the thioredoxin/GCRG213 fusion protein was expressed in E.coli. The product was obtained by means of direct purification from a denaturing polyacrylamide gel. Results SDS-PAGE analysis showed the thioredoxin/GCRG213 fusion protein with relative molecule mass of 29.4kDa was highly expressed. The thin layer gel scanning analysis showed that the yield of GCRG213 fusion protein was 28.7% of the total bacterial protein. The product was obtained with a purity of about 100% by means of direct purification from a denaturing polyacrylamide gel. Conclusion The thioredoxin/GCRG213 fusion protein was successfully expressed in E.coli and the product with high purity was obtained, which laid the foundation for the function research and antibody preparation hereafter.
ABSTRACT
Objective To prepare a polyclonal antibody against gastric cancer-related protein GCRG224.Methods The thioredoxin/GCRG224 fusion protein was expressed in E.coli.A polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein.The titer and specificity of the antibody were determined by ELISA and Western-blot,respectively.Results The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8kD was over-expressed in E.coli.The purity of expressed products directly purified from a denaturing polyacrylamide gel was about 100%.The polyclonal antibody against GCRG224 was obtained.The ELISA titer of antiserum against GCRG224 was about 1∶256 000.Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically.Conclusion The polyclonal antibody against GCRG224 has been successfully prepared,which lays the foundation for further study on the biological function and the possible role of the GCRG224 in the development of gastric carcinoma.