中国生物工程杂志

15-hydroxyproglandin dehydrogenase(PGDH) is a tumor suppressor gene. PGDH transcript and protein are both highly expressed by normal tissue but are nearly undetectable in several tumors. Total RNA was isolated from human normal colonic epithelia. Then the PGDH cDNA gene was amplified by reverse transcript polymerase chain reaction (RT-PCR). The amplified fragment was orientationly linked into the prokaryotic expression vector pBV220 by T4 DNA ligase and then sequenced. The result indicated that the cloned gene was a corrected PGDH. Then the recombined expression plasmid was transduced into DH5a by TSS, and the expression of PGDH protein was induced by temperature for 3 hours at 42 C, and the fusion protein of PGDH-His6 was analyzed by SDS-PAGE and Western blot after lysis of bacteria. The SDS-PAGE result showed that cloned recombinant protein was expressed in the inclusion body with molecular weight of approximated 29kDa. The PGDH fusion protein containing about 30% of total somatic protein, was stably expressed in E.coli. After ultrasonication and purification by immobilized metal chelation affinity chromatography, homogenous protein was obtained and reached 95% purity as proved by SDS-PAGE. Western blot identified its specificity. The expressed PGDH protein had highly bioactivity, and its enzyme activity is about 3.7×104U/mg.