人激肽释放酶基因克隆及真核表达的实验研究

Cloning of Human Tissue Kallikrein Gene and Expressing it in CHO Cell

为构建人激肽释放酶（human kallikrein,hk）基因真核表达细胞系,从人胎胰中用RT-PCR方法扩增了hk基因全序列及其前导肽序列,克隆到真核表达质粒pCDNA3.1中,得到含全长hk cDNA及其前导肽序列的真核表达质粒pCDNA3-hk,并用脂质体lipofectamine介导其转化到野生型CHO细胞株中,获得了能稳定表达hk蛋白的CHO细胞系CHO-hk。检测CHO细胞及其培养基中hk活性水平及培养基对兔血压的影响。结果表明,97％的hk分泌到细胞外,其中62％左右以活性形式存在,另38％以酶原形式存在。培养基浓缩液具有显著降血压作用。说明CHO-hk细胞系能分泌具完整生物活性的hk,为后续生产治疗高血压的基因工程药物打下了基础。

To construct the eukaryotic expressing cell line of human kallikrein, human kallikrein gene sequence inducing its leader peptide sequence was amplified by RT-PCR from human fetus spleen. The whole sequence was lin-gated into a eukaryotic expressing plasmid pCDNA3.1 to form the positive clone named pCDNA3. 1-hk. Then pCD-NA3.1-hk was introduced by lipofectamine into wide type CHO cells . Human kallikrein activity in the cells and in the medium was detected, the effect of medium on the blood pressure of rabbit was also assayed. The eukaryotic expressing plasmid includling the intact hk cDNA and its leader peptide sequence was obtained, as well as the CHO cell line which expresses human kallikrein protein enduringly. 98% of the kallikrein protein was secreted into the medium, among them 62% is in the active form ,and the other 38% is in the preenzyme form. The concentrated medium obver-sly reduce the blood pressure of rabbit. Human kallikrein with intact activity can be secreted by CHO-hk cell line, and this make a good ground to product gene engineering medicine to treat hypertension.