摘要
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。
Objective To achieve high-level secretory expression of recombinant human pepsinogenⅠ（PGⅠ）in Hansenula polymorpha and prepare the purified recombinant PG Ⅰ into a calibrator for in vitro diagnostic reagents. Methods According to the H. polymorpha-prefered codon, PG Ⅰ gene was designed, synthesized and cloned into expression vector p RMHP2. 1. The constructed recombinant plasmid was transformed to H. polymorpha 26012 by electroporation for several rounds of passage and stabilization, and the strain in which PG Ⅰwas highly expressed in a secretory form was screened and identified for the integrated site and copy number of heterologous gene. Fermentation liquid was prepared in a 200 L fermenter, from which recombinant PG Ⅰprotein was purified by nickel ion affinity chromatography and determined by a latex-enhanced immunoturbidimetric kit. The purified PG Ⅰprotein was prepared into calibrators at concentrations of 100 and 50 μg / L respectively and tested for stability. Results Restriction analysis and DNA sequencing proved that recombinant plasmid p RMHP2. 1-PGⅠwas constructed correctly. After rounds of screening, a recombinant H. polymorpha strain was obtained, in which PG Ⅰ protein with a relative molecular mass of about 45 000 was expressed. The yield of expressed product was more than 100 mg / L, while PG Ⅰ gene of was integrated into the host r DNA site, and the gene copy number was not less than 20. The purity of purified recombinant PG Ⅰ protein was 94. 5%. The mean degradation limit of active ingredient contents of PGⅠ as a calibrator in accelerated stability, real time stability and on-board stability tests were not more than 10%. Conclusion Recombinant PG Ⅰ protein was highly expressed in a secretory from in H. polymorpha, which showed high stability and might be used as a calibrator for in vitro diagnostic reagents.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第2期125-132,共8页
Chinese Journal of Biologicals
关键词
胃蛋白酶原Ⅰ
汉逊酵母表达系统
分泌表达
纯化
PepsinogenⅠ（PGⅠ）
Hansenula polymorpha expression system
Secretory expression
Purification
