摘要
目的:探索一种高效制备人胃蛋白酶原Ⅱ(pepsiongenⅡ,PGⅡ)体外诊断试剂用校准品的方法。方法:以汉逊酵母ATCC26012作为宿主菌,以携带Zeocin及G418双重筛选标记的pRMHP2.1质粒为载体,以遗传密码子优化设计后的PGⅡ序列为目的基因,通过电转化及后续多轮次的传代与稳定,筛选获得高水平分泌表达PGII蛋白的重组菌株26012/PGⅡ。采用Ni柱亲和层析的方法从200L发酵罐大量制备的培养液中纯化出高纯度的重组PGⅡ蛋白,将该蛋白质进行校准定值后配制成6种不同浓度的成套PGⅡ校准品,并对该系列校准品的实时稳定性、加速破坏性及开瓶稳定性展开评价。结果:筛选获得的重组汉逊酵母菌株26012/PGⅡ外源基因整合拷贝数高于40个且整合于染色体的r DNA位置,该菌株以分泌形式表达重组PGⅡ蛋白,表达量超过50mg/L,发酵培养液经Ni柱亲和层析纯化后,重组PGⅡ蛋白纯度达93.8%。通过免疫比浊试剂盒定量检测,该PGⅡ蛋白的活性定值与实际蛋白质量之间的比值达0.85,配制后的PGⅡ校准品在4℃实时保存1年、37℃加速破坏2周及4℃开盖保存2周后,校准品定值的平均下降幅度都不超过10%,其稳定性能不逊色于商品化的校准品。结论:汉逊酵母重组表达的PGⅡ蛋白可用作体外诊断试剂的校准品。
Objective： To efficiently produce the recombinant PG Ⅱ calibrators for in vitro diagnostic reagents. Methods： The PGⅡ gene designed with the H. polymorpha-prefered codon was cloned into expression vector pRMHP2.1 carrying Zeocin and G418 dual screening markers. After electroporation and rounds of passage and stabilization,the recombinant high-level secretory expression strain 26012 / PGⅡwas screened. Furthermore,the recombinant PGⅡ protein was purified using Ni-affinity chromatography from the fed-batch cultures in a 200 L fermenter. After determined by a latex-enhanced immunoturbidimetric kit,the purified PGⅡ protein was diluted into serial calibrators with 6 different concentrations and the real-time stability,accelerated stability and on-board stability testing was conducted. Results： The screened recombinant expression strain 26012 / PGⅡ,with the PGⅡ gene integrated into the host r DNA site and the gene copy number not less than 40,yield more than 50 mg /L PGⅡ of cultures in a secretory form. The purity of the purified recombinant PGⅡ protein was 93. 8% using Niaffinity chromatography from the fed-batch cultures,and the ratio of protein concentration determined using the immunoturbidimetric kit to that using BCA method was about 0. 85. After diluted into serial calibrators,the general degradation limit of 10% active content was observed in a1-year real-time stability testing,or a 2-week saccelerated stability testing,or a 2-weekson-board stability testing. Furthermore,it was also shown that the stability of purified recombinant PG Ⅱ was not inferior to that of commercial PG Ⅱ calibrators. Conclusions：Recombinant PGⅡ protein secreted from H. polymorpha is suitable for use as the PG Ⅱ calibrators for in vitro diagnostic reagents.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2016年第9期38-46,共9页
China Biotechnology
关键词
胃蛋白酶原Ⅱ
汉逊酵母
分泌表达
纯化
校准品
Pepsinogen Ⅱ
Hansenula polymorpha
Secretory expression
Purification
Calibrator
