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

摘要:

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11 mg/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。

Abstract:

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.