Cloning and expression of extracellular domain of prostate specific membrane antigen in Escherichia coli and preparation of polyclonal antibody.
- Author:
Chuan-Zhong YE
1
;
Xu-Dong ZHAO
;
Fang-Lin ZHANG
;
Zhen LIN
;
Ming XU
;
Yong-Kang ZHANG
;
Chang-Qing CHEN
Author Information
1. Department of Urology, Zhongshan Hospital, Medical Center of Fudan University, Shanghai 200032, China. chuanzhong@mycity.com.cn
- Publication Type:Journal Article
- MeSH:
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
- From:
Chinese Journal of Biotechnology
2002;18(1):35-39
- CountryChina
- Language:Chinese
-
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.
