tTSF基因的酵母表达及其基因治疗研究

目的 选择PF4，TSP1的高活性片段及肿瘤细胞靶向肽，设计合成融合基因—tTSF，进行酵母表达及小鼠基因治疗研究。方法 根据PF4和TSP1的抑制血管生成高活性片段设计合成TSF基因，并在N-端接上肿瘤细胞靶向肽，设计合成了tTSF。将tTSF基因构建到pPIC9质粒，重组质粒pPIC9／tTSF转化GSll5酵母菌株进行表达，并检测表达产物对血管内皮细胞生长的抑制作用。将tTSF基因克隆到pcDNA3．1（＋）载体，重组质粒pcDNA3．1（＋）／tTSF通过皮下注射治疗C57BL／6小鼠移植肿瘤Lewis肺癌。结果 重组质粒pPIC9／tTSF转化GSll5酵母菌株后，获得了tTSF的高效表达，表达产物对血管内皮细胞生长产生明显的抑制作用；皮下注射重组质粒pcDNA3．1（＋）／tTSF后，C57BL／6小鼠移植肿瘤Lewis肺癌的生长受到明显抑制。结论 tTSF基因设计成功，其酵母表达产物对血管内皮细胞生长产生具有明显的抑制作用，tTSF基因的体内基因治疗也具有显著的抗肿瘤效应。

tTSF gene expression in P. Pastoris and its gene therapy on tumor growth

Objective To detect the gene therapy of tTSF on mice and the inhibitory effect of tTSF peptide expressed in P. Pastoris on endothelial cells which was designed from PF4(58-70) ,TSP(429-459) and a tumor-targeted fragment. Methods tTSF gene was designed, containing the fragments of PF4(58-70), TSP1 (429-459) and a tumor-targeted fragment which was ligated with Gly bridge. The tTSF gene was cloned into pcDNA3. 1(+) and pPIC9 plasmids,respectively. The recombined plasmid pPIC9/tTSF was transformed into P. Pastoris,which secreted tTSF peptide after induced with methanol. With the method of MTT assay,the effects of tTSF peptide on the growth of endothelial cells were detected. The recombined plasmid pcDNA3. 1 (+)/tTSF was injected into C57BL/6 mice to detecte the inhibitory effect on the growth of implanted lewis lung cancer. Results tTSF peptide with molecular weight of 7kDa was produced in P. Pastoris after induced with methanol. Which inhibited the growth of endothelial cells significantly. The recombined plasmid pcDNA3. 1 (+)/tTSF showed significant inhibitory effect on the growth of lewis lung cancer in vivo. Conclusions The design of the tTSF gene was successful. tTSF peptide had significant inhibitory effect on angiogenesis in vitro. The tTSF gene therapy also showed potent inhibitory effect on the growth of cancer in vivo.