陈丹;李璐;班靖洋;郭玉争;王维龙;刘娟;李鼎锋;刘勇;

• 1:北京安百胜生物科技有限公司

摘要(Abstract)：

目的实现人胃蛋白酶原Ⅰ(pepsiongenⅠ,PGⅠ)在汉逊酵母表达系统中的高效分泌表达,并将纯化的重组PGⅠ蛋白制备成体外诊断试剂用校准品。方法根据汉逊酵母遗传密码子偏爱性优化设计并合成PGⅠ基因,克隆至表达载体p RMHP2.1中,电转化汉逊酵母宿主菌26012,进行多次传代及稳定,筛选高水平分泌表达PGⅠ的菌株,并对该菌株的目的基因整合位置及整合拷贝数进行检测。应用200 L发酵罐大规模制备发酵培养液,通过Ni柱亲和层析方法纯化获得重组PGⅠ蛋白,经胶乳增强免疫比浊试剂盒进行PGⅠ蛋白的定值,应用类血清基质液制备100及50μg/L浓度的PGⅠ校准品后检测稳定性。结果重组表达质粒p RMHP2.1-PGⅠ经双酶切及测序鉴定证明构建正确。筛选获得的重组汉逊酵母菌株分泌表达的PGⅠ蛋白相对分子质量约45 000,表达量达100 mg/L以上,其外源基因整合于宿主r DNA位置,整合拷贝数不低于20个。纯化后的重组PGⅠ蛋白纯度为94.5%,配制后的PGⅠ校准品经4℃实时保存稳定性、37℃加速破坏性及4℃开盖保存稳定性试验,校准品定值的平均下降幅度均不超过10%。结论应用汉逊酵母表达系统可高效分泌PGⅠ蛋白,该蛋白具有良好的稳定性,可用作体外诊断试剂的PGⅠ校准品。

关键词(KeyWords)： 胃蛋白酶原Ⅰ;汉逊酵母表达系统;分泌表达;纯化

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 陈丹;李璐;班靖洋;郭玉争;王维龙;刘娟;李鼎锋;刘勇;

Email:

DOI: 10.13200/j.cnki.cjb.001651

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