TGFα-PE 40的基因克隆、表达和对人癌细胞生长的抑制作用

GENE CLONING,EXPRESSION OF FUSION PROTEIN TGFα-PE 40 AND ITS INHIBITION ACTIVITY ON CANCER CELL GROWTH

本文报告了构建人转化生长因子α和绿脓杆菌外毒素融合基因的表达型重组质粒pYX382和pYX 3825,两种质粒转化大肠杆菌BL21后,经IPTG 诱导,表达融合蛋白TGFα-PE 40,表达产物分子量为46 kd,具有和抗TGF a抗体和抗PE 抗体反应的能力,重组质粒pYX 3825带有信号肽顺序,因而在细菌中表达产物大部分被分泌到培基和周质中。TGFa-PE 40具有和细胞表面EGFR 结合的能力,因而,能与过度表达EGFR 的癌细胞结合,将毒素蛋白的活性部分带入细胞而显示对癌细胞蛋白质合成的强烈抑制和对癌细胞的杀伤。

Recombinant plasmid pYX 382 andpYX 3825 were constructed by fusing thecDNA encoding transforming growth fac-tor type alpha(TGFα)to Pseudomonasexotoxin gene(PE)in which the cell re-cognition domain was deleted.The chime-ric proteins produced by host E.Coli cel-ls BL 21 transformed by plasmid pYX 382and pYX 3825 are termed TGFα-PE 40which reacts with antibody against TGF αor antibody against PE in immunoblottingto show a 46kd protein band reflectingthe fusion of 56 kD TGF α peptide and40 kD truncated Pseudomonas exotoxinmolecule.An additional signal sequenceOmpA was inserted into upstream regionof TGFα cDNA in plasmid pYX 3825 re-sulting in the partly secreting of expres-sion product into medium and periplasmof the cells.TGF α-PE 40 was purifiedfrom medium by MONO Q ion exchangecolumn and TSK 250 gel filtration columnattached to Pharmacia EPLC system.TheTGFα-PE 40 molecules showed a very st-rong activities inhibiting the protein syn-thesis and killing the cancer cells overex-pressing EGF receptor on the cell surface.