[Cloning and expression of extracellular domain of prostate specific membrane antigen in Escherichia coli and preparation of polyclonal antibody].

Abstract

Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR. The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x. Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported. The edPSMA was expressed in E. coli as part of a fusion protein with MBP as the induction of IPTG. Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5. MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120 kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody. The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue.

• Authors: Chuan-Zhong Ye, Xu-Dong Zhao, Fang-Lin Zhang, Zhen Lin, Ming Xu, Yong-Kang Zhang, Chang-Qing Chen
• Journal: Sheng wu gong cheng xue bao = Chinese journal of biotechnology (Sheng Wu Gong Cheng Xue Bao) Vol. 18 Issue 1 Pg. 35-9 (Jan 2002) ISSN: 1000-3061 [Print] China
• PMID: 11977596 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies
• Antigens, Surface
• DNA, Complementary
• Recombinant Fusion Proteins
• Carboxypeptidases
• FOLH1 protein, human
• Glutamate Carboxypeptidase II

Topics

• Animals
• Antibodies (immunology)
• Antibody Formation
• Antigens, Surface
• Carboxypeptidases (biosynthesis, genetics, immunology, isolation & purification)
• Chromatography, Affinity (methods)
• Cloning, Molecular
• DNA, Complementary (genetics)
• Escherichia coli (genetics)
• Gene Expression
• Genetic Vectors
• Glutamate Carboxypeptidase II
• Humans
• Mice
• Mice, Inbred BALB C
• Protein Structure, Tertiary (genetics, physiology)
• Recombinant Fusion Proteins (biosynthesis, genetics, immunology, isolation & purification)
• Reverse Transcriptase Polymerase Chain Reaction (instrumentation)