生长激素释放激素和人血清白蛋白融合蛋白的克隆表达  

Cloning and Expressing of HSA-GHRH Fusion Protein

作　　者：王楠[1] 赵洪亮[1] 刘志敏[1] 刘党生[2] 薛冲[1] 熊向华[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学制药工程学院,辽宁沈阳110015

出　　处：《生物技术通讯》2007年第4期574-577,共4页Letters in Biotechnology

摘　　要：目的:通过与人血清白蛋白(HSA)融合,延长生长激素释放激素(GHRH)在体内的半衰期。方法:根据毕赤酵母偏爱密码子重新设计GHRH的核酸序列,并通过化学合成和重叠PCR法将GHRH的N端与HSA的C端通过一个11肽的接头连接,获得GHRH和HSA融合的全长基因序列。构建pPIC9-HSA-GHRH表达载体,电击转化毕赤酵母GS115感受态细胞,通过表型筛选和诱导表达实验得到蛋白表达工程菌,对表达产物进行分离纯化和生物学活性分析。结果:克隆了HSA-GHRH融合基因,构建了pPIC9-HSA-GHRH融合表达载体;电击转化后通过表型筛选和诱导表达实验得到蛋白表达工程菌;经分离纯化后,对表达产物的生物学活性分析显示其在体内有促进生长的作用。结论:与人血清白蛋白的融合有效地提高了GHRH的表达水平,并延长了GHRH的半衰期。Objective： To prolong the half-life of growth hormone releasing hormone（GHRH） by constucting human serum albumin（HSA）-GHRH fusion protein. Methods： The nucleic acid sequence of GHRH was redesigned according to Pichia pastoris bias codon. N-terminal of GHRH and C-terminal of HSA was linked through a eleven-peptides spacer. This HSA-GHRH fusion gene was obtained by chemical synthesis and overlap extension PCR. Then the expression vector of pPIC9-HSA-GHRH was constructed and transformed into P.pastoris by electroporation. Through phenotype selection and inducing assay, the expression engineering strain was obtained. Finally, the expressed products was purified and its bioactivity was analysised. Results： The fusion gene was cloned and the vector was constructed. Through phenotype selection and inducing assay, the expression engineering strain was obtained. And the bioactivity analysis experiment showed that the fusion protein has the activity of growth promotion in vivo. Conclusion： The expression level of GHRH was increased and its half-life was prolonged by fusing to HSA.

关 键 词：生长激素释放激素 人血清白蛋白 毕赤酵母 表达 生物学活性 

分 类 号：Q78[生物学—分子生物学]