牛透明质酸酶PH-20核心区在毕赤酵母中的高效表达

摘要

Objective To express the core domain coding sequence of hyaluronidase PH-20 from bull testis in Pichia pastoris, and to achieve the recombinant expression of PH-20 protein. Methods The core domain coding sequence of PH-20 without signal and anchor sequences was optimized according to the codon usage preference of P. pastoris, ligated with plasmid pPICZaA and transformed into P. pastoris SMD1168H. After induced by 0.5%(v/v)methanol for 72 h, the hyaluronidase activity in the supernatant was determined using 3, 5-dinitrosalicylic acid (DNS) method. Results Recombinant yeast strain which contained the core domain coding sequence of PH-20 was induced by 0.5%(v/v)methanol for 72 h, and the hyaluronidase activity in the supernatant was up to 135.2 U/mL. Conclusion PH-20 from bull testis can be high-levelly expressed in P. pastoris.%目的利用毕赤酵母表达牛源透明质酸酶核心区，以实现透明质酸酶的重组制备。方法将去除信号肽和锚定区域的牛源透明质酸酶PH-20成熟肽编码序列按照毕赤酵母密码子偏爱性进行优化，然后连入pPICZaA表达载体并转入毕赤酵母SMD1168H菌株。0.5%（v/v）甲醇诱导表达72 h后采用3,5-二硝基水杨酸（3,5-dinitrosalicylic acid，DNS）法测定培养液上清中透明质酸酶活性。结果包含PH-20核心序列（1332 bp）的重组酵母经甲醇诱导表达72 h，培养液上清中透明质酸酶活性达135.2 U/mL。结论实现了牛源透明质酸酶基因在毕赤酵母中的高效表达。