摘要
目的 :构建和表达一种抗菌肽和人酸性成纤维细胞生长因子的融合蛋白 ,在创伤皮肤表面应用时能裂解成有功能的抗菌肽和人酸性成纤维细胞生长因子 ,预防伤口感染和促进皮肤修复。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基因和人酸性成纤维细胞生长因子基因。再将两者作为模板 ,用PCR方法扩增出编码融合蛋白的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 96h ,SDS PAGE电泳检测融合蛋白的表达 ,并用Western blot杂交检测融合蛋白的免疫原性。利用肝素亲和层析纯化融合蛋白 ,同时检测融合蛋白的抑菌和促3T3Bal/b细胞分裂活性。结果 :筛选出能表达相对分子质量约为 19kD融合蛋白的重组转化株 ,SDS PAGE电泳检测显示甲醇诱导 96h后 ,融合蛋白表达量达到最高 ,且能与人酸性成纤维细胞生长因子抗体产生免疫反应。融合蛋白没有抑菌活性 ,而裂解产物则具有抑菌活性。此外 ,融合蛋白的促细胞分裂活性与野生型人酸性成纤维细胞生长因子相比没有明显的降低。结论 :获得含凝血酶切割位点的抗菌肽和人酸性成纤维细胞生长因子的融合蛋白 ,并在毕赤酵母中实现了表达。融合蛋白的裂解产物具有抑菌活性。
AIM: To construct a new-type fusion protein composed of cecropin AD and acidic fibroblast growth factor with the site of thrombin digested,which can cleave into cecropin AD and acidic fibroblast growth factor with thrombin secreted from wound when used in wounded skin.METHOD:The DNA that coded fusion protein was cloned into vector pPICZαA and transformed into Pichia pastoris SMD1168.The expression of the fusion protein was detected by 15% SDS-PAGE and western-blot.The fusion protein was purified by heparin affinity,and its suppression on bacterial and mitogenic was detected.RESULT: After inducing with methanol for 96 h,the expression level reached the highest.On the other hand,the fusion protein could not restrain the growth of E.coli K_ 12D_ 31,but it still could stimulate the proliferation of the 3T3Bal/b cells.The production of fusion protein split by thrombin can restrain the growth of E.coli K_ 12D_ 31.CONCLUSION:The fusion protein cecropin AD and acidic fibroblast growth factor is expressed in Pichia pastoris successfully.The production of fusion protein split by thrombin can restrain the growth of E.coli and stimulate the proliferation of the 3T3Bal/b cells.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2005年第1期63-68,共6页
Journal of China Pharmaceutical University
基金
国家十五"863"基金资助项目 (No .2 0 0 2AA2Z3 3 18
2 0 0 1AA2 15 13 1)
国家"973"基金资助项目 (No .G19990 5 42 0 4)
广东省自然科学基金资助项目 (No .0 10 42 4)~~