【摘要】 目的:为了延长人生长激素(HGH)在血浆中的半衰期,构建了高效分泌表达人血清白蛋白(HSA)与HGH融合蛋白(HSA-HGH)的工程毕赤酵母菌株。方法:从人胎肝cDNA文库中扩增HSA基因,从HGH工程菌载体中扩增HGH基因,将其克隆至真核表达载体pHIL-D2,载体线性化后采用电击法转化毕赤酵母GS115。通过原位双层膜法筛选高效分泌表达菌株。分别采用SDS-PAGE和Western印迹鉴定融合蛋白。对工程酵母培养条件进行研究,发酵液经离子交换层析、亲和层析和分子筛层析纯化。纯化的蛋白经N端序列测定,分子量测定和等电聚焦电泳进行鉴定。冻干的蛋白制剂经食蟹猴试验进行药代动力学和药效动力学测试。结果:确立了工程酵母的最佳培养工艺,融合蛋白表达量达100mg/L。纯化后蛋白纯度达95%以上,得率达42%。融合蛋白与预期结果一致,经食蟹猴试验,显示有良好的生物活性,与等摩尔剂量的重组人生长激素相比,半衰期延长6.8倍,清除率慢44倍。结论:融合蛋白呈现明显的长效动力学特征,为开发重组长效人生长激素HSA-HGH融合蛋白药物(rHSA-HGH)奠定了基础。更多还原

【Abstract】 Objective: To prolong the half-life of human growth hormone(HGH) in the blood plasma,the engineering Pichia pastoris yeast strain of efficient expression and secretion of human serum albumin(HSA)-HGH fusion protein was constructed.Method: The genes of HSA and HGH were amplified respectively from human fetal liver cDNA library and HGH engineering strain vector.After cloned into the expression vector pHIL-D2,the recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation.The efficient expression and secreting strain was screened out by in situ double filter screening method,and then confirmed respectively by SDS-PAGE and Western blotting.Its culture condition was researched.The fusion protein was purified from the culture of the yeast by ion exchange,affinity and gel filtration chromatography,and then confirmed by N terminal sequence test,molecular weight test and isoelectric focus electrophoresis.Finally,the pharmacokinetics and pharamacodynamics test of frozen dry protein powder were carried out in cynomolgus monkeys.Results: The best culture condition of engineering yeast was established,with the highest yield of 100 mg/L.The purity was over 95 percent,and the productivity was over 42 percent.The protein demonstrated good bioactivity by the cynomolgus mokeys test,and has a 6.8-fold longer half-life and a 44-fold slower clearance than equimolar doses of HGH.Conclusion: The fusion protein demonstrates a long acting characteristic,which suggests its promising application in clinical medicine.更多还原