胱抑素C在巴斯德毕赤酵母的高效分泌表达及免疫原性分析

HIGH EXPRESSION OF CYSTATINE C IN PICHIA PASTORIS AND ITS IMMUNOGENICITY

目的根据巴斯德毕赤酵母(P.pastoris)密码子的偏好,将人胱抑素C(CysC)基因进行密码子优化,在P.pastoris中表达CysC,并对重组蛋白进行生物学活性评价。方法按照P.pastoris表达系统密码子偏好,优化基因序列,人工合成完整的基因;构建pPIC9k-CysC重组表达质粒,用电穿孔法转化P.pastoris GS115,在MD平板上筛选重组克隆,用G418快速筛选高拷贝转化子,阳性克隆经甲醇诱导表达后,取培养物上清液,经SDS-PAGE电泳和颗粒增强透射免疫比浊法鉴定表达情况。经Western blot和动物实验鉴定重组蛋白的免疫原性。结果表达产物相对分子质量约19 000,优化后的人CysC基因在P.pastoris转化菌得到了高效表达,表达量占分泌总蛋白的80%以上,产物浓度为600～950mg/L。Western blot和动物实验显示重组蛋白具有较好的免疫原性。结论密码子优化后CysC在P.pastoris中获得高效分泌表达,可用于制备抗血清和诊断试剂标准品。

E7 Objective According to codom preference of P. pastoris, the human cystatine C （CysC） protein was opti- mized and expressed in P. pastoris and the recombination protein was assessed for its biologic activity （BA）. Methods According to preference of P. pastoris expression system, the gene sequence was optimized, a complete gene was then synthesized. A recombi- nant expression plasmid pPlC9k-CysC was constructed, and pastoris GSl15 was transformed by electroporation. The recombinant clone was screened on the MD plate, the high copy transformants were fast screened by using G418. After methanol-induced ex- pression, supernate of the culture was collected to identify the expression by using SDS-PAGE and PETIA. The immunogenicity of recombinant protein was idedified by Western blot and an animal experiment. Results The relative molecular mass of the expres sion product was 19 000, the optimized human CysC gene was highly expressed in P. pastoris, its expression volume accounted for more than 80% of the excretion of the total protein, with the concentration of 600-950 mg/L. Western blot and the animal experi- ment showed the recombinant protein had a better immunogenicity. performance secretion expression in P. pastoris, which can be used as reagent. Conclusion The optimized CysC gene demonstrates high- a standard preparation for preparing antiserum and diagnostic