摘要
目的 :构建和表达一种带有凝血酶切割位点的天蚕素杂合肽AD和人酸性成纤维细胞生长因子 (acidicfibroblastgrowthfactor ,aFGF)突变体的融合蛋白S ,在创伤皮肤表面应用时能自动裂解成有抗感染功能的天蚕素杂合肽AD和皮肤修复功能的aFGF突变体。方法 :利用PCR技术分别扩增出带有凝血酶切割位点的天蚕素杂合肽AD基因和aFGF突变体基因。再利用两者作为模板 ,用PCR方法扩增出编码融合蛋白S的DNA。将该DNA片段插入到载体pPICZαA中 ,并利用电转法将质粒转入毕赤酵母smd116 8中 ,Zeocin抗性筛选重组转化子。甲醇诱导 72h ,SDS -PAGE电泳检测融合蛋白S的表达 ,并用Western -blot杂交检测融合蛋白S的免疫原性。结果 :筛选出的重组转化株在甲醇诱导后能表达相对分子质量约为 190 0 0的融合蛋白S ,而该蛋白能与aFGF抗体产生免疫反应。结论 :用PCR方法获得编码含凝血酶切割位点的天蚕素杂合肽AD和aFGF突变体的融合蛋白SDNA 。
A new-type fusion protein S composed of cecropin AD and acidic fibroblast growth factor mutant was constructed and expressed in Pichia pastoris. It can cleave between cecropin AD and acidic fibroblast growth factor mutant automatically when it was used in wounded skin, then can prevent the wound from bacteria and repair the skin. Methods: The DNA that coded fusion protein S was cloned into vector pPICZαA, and transfered to Pichia pastoris smd1168. After induced 72 h with methanol, the expression of the fusion protein was detected by 15% SDS-PAGE and Western-blot. The fusion protein S was purified by heparin affinity. Results: The fusion protein S was expressed in Pichia pastoris induced by methanol and had antigen specificity with aFGF antibody. Conclusion:The fusion protein S that can cleave between cecropin AD and acidic fibroblast growth factor automatically when it was used in wounded skin was expressed in Pichia pastoris successfully.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
2004年第4期420-425,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家十五"86 3"基金资助项目 (2002AA2Z3318
2 0 0 1AA2 15 131)
国家"973"基金资助项目 (G19990 5 4 2 0 4 )
