人胰岛素前体在毕赤酵母中的组成型表达及其活性

Expression of human insulin precursor in Pichia pastoris using GAP promoter and activity of expressed product

目的通过毕赤酵母GAP启动子高效分泌表达人胰岛素前体,并检测重组胰岛素类似物的生物活性。方法经密码子优化并人工合成人胰岛素前体基因IS基因,插入pGAPZaA组成型表达载体中,构建重组表达质粒pGAPZaIS,电转化至感受态毕赤酵母SM1168菌株中,PCR鉴定阳性重组子;高抗性YPD平板筛选高蛋白表达株并优化表达条件;表达产物经Tricine-SDS-PAGE和Western blot分析;用吸附树脂吸附、阳离子交换层析、超滤纯化后,用经TPCK处理的胰蛋白酶切除人工C肽,经阴离子交换层析和醋酸锌沉淀纯化后,进行Tricine-SDS-PAGE分析;将12只链脲佐菌素(streptozotocin,STZ)致糖尿病模型的小鼠随机分为A组和B组,A组小鼠经腹腔注射含1.8%甘油的注射用水作为对照组,B组小鼠经腹腔注射用胰岛素类似物配制的0.05 mg/ml注射用水剂(含1.8%甘油),10μl/g,空腹6 h后,断尾取血,用血糖仪检测小鼠血糖值。结果重组表达质粒pGAPZa-IS经双酶切和测序鉴定,证明构建正确;共获得5株高抗性重组菌,其表达产物均可见相对分子质量约6 000的目的蛋白条带,与小鼠抗胰岛素单克隆抗体不发生特异性反应,5号菌72 h表达量最高;C肽切除后,获得了纯胰岛素类似物;模型小鼠在注射胰岛素类似物6 h后,血糖值均明显降低,B组小鼠注射前后血糖值及A、B组间的血糖值差异均有统计学意义(P<0.01)。结论在毕赤酵母中,利用GAP启动子成功高效表达了胰岛素前体,并具有良好的降血糖活性。

Objective To express human insulin precursor in Pichia pastoris using glyceraldehyde-3-phosphate dehydrogenase promoter （pGAP）, and determine the biological activity of recombinant insulin analogue. Methods Human insulin precursor IS gene was synthesized using P. pastoris preferred codons and inserted into vector pGAPZaA. The constructed recombinant plasmid pGAPZa-IS was transformed to competent P. pastoris SMD1168 strain by eleetroporation, and the positive recombinants were identified by PCR. The high protein-expressing strains were screened by highly zeocinresistant YPD plate, and the condition for expression was optimized. The expressed product was identified by Tricine-SDS- PAGE and Western blot, adsorbed by adsorption resin, purified by cation exchange chromatography and uhrafihration, then further purified by anion exchange chromatography and zinc acetate precipitation after removal of synthetic C pep- tide with TPCK-treated trypsin, and analyzed by Trieine-SDS-PAGE. Twelve diabetic mice caused by streptozotocin （STZ） were divided into control （A） and test （B） groups. The mice in group A were injected i. p. with water for injection, containing 1.8% glycerol, while those in group B with 0. 05/ml insulin analogue solution formulated with water for injection, containing 1. 8% glycerol, each at a dosage of 10 μ l/ g bodyweight. After a fast of 6 h, the mice were determined for blood sugar. Results Restriction analysis and sequencing proved that recombinant plasmid pGAPZa-IS was constructed correctly. Five high zeocin-resistant recombinant strains were screened, in which the expressed product, with a relative molecular mass of about 6 000, showed no specific reaction with moue monoclonal antibody against insulin. The expression level in recombinant strain 5 72 h after culture was the highest in five strains. Pure insulin analogue was obtained after removal of C peptide. The blood sugar level of diabetic mice decreased significantly 6 h after iniection with insulin analogue, whichshowed significant difference in group B before and after injection and in groups A and B （P 〈 0. 01 ）. Conclusion Insulin precursor was successfully expressed in P. pastoris by using GAP promoter, which showed high hypoglycemic activity.