hK-Fc融合蛋白的改良、表达及其生物活性的分析

Modification,expression and bioactivity analysis of hK-Fc fusion protein

为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5～10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。

To prolong serum half-life of human kallikrein（hK） and enhance its secretion rate,we modified hK gene and constructed a new form of recombinant hK protein（hK＇-Fc）.We amplified hK gene and Fc sequence,replaced the signal peptide of hK gene with murine signal peptide,constructed native expression plasmid of pcDNA-hK-Fc and modified expression plasmid of pcDNA-hK＇-Fc,then transfected to CHO cells respectively.After the stable cell lines were screened,we compared the secretion rate between native fusion protein and modified fusion protein,purified fusion protein through Protein A＋G affinity chromatography column and investigated the bioactivity of fusion protein.The results showed that recombinant vectors encoding fusion protein hK-Fc and hK＇-Fc were constructed successfully;CHO cell lines stably secreting fusion protein were obtained,the yield is higher than 11 mg/L;Secretion rate was enhanced by 5-10 times after the signal peptide of fusion protein was modified;Fusion protein has enzymatic activity in vitro.The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy.