汉逊酵母重组表达的人胃蛋白酶原Ⅱ校准品研制

doi:10.13523/j.cb.20160905

中国生物工程杂志

2016, Vol. 36

Issue (9): 38-46 DOI: 10.13523/j.cb.20160905

技术与方法

汉逊酵母重组表达的人胃蛋白酶原Ⅱ校准品研制

陈丹, 郭玉争, 班靖洋, 李璐, 王维龙, 李鼎锋, 刘勇

北京安百胜生物科技有限公司北京 100176

Development of Recombinant Human PepsinogenⅡ Calibrators Derived from Hansenula polymorpha

CHEN Dan, GUO Yu-zheng, BAN Jing-yang, LI Lu, WANG Wei-long, LI Ding-feng, LIU Yong

Abzymo Biosciences Co. Ltd., Beijing 100176, China

全文: PDF(780 KB) HTML

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rDNA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。

关键词： 纯化; 胃蛋白酶原Ⅱ; 汉逊酵母; 校准品; 分泌表达

Abstract:

Objective:To efficiently produce the recombinant PGⅡ 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 200L 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 rDNA site and the gene copy number not less than 40, yield more than 50mg/L PGⅡ of cultures in a secretory form. The purity of the purified recombinant PGⅡ protein was 93.8% using Ni-affinity 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 PGⅡ was not inferior to that of commercial PGⅡcalibrators. Conclusions:Recombinant PGⅡ protein secreted from H. polymorpha is suitable for use as the PGⅡ calibrators for in vitro diagnostic reagents.

Key words: Secretory expression Pepsinogen Ⅱ Purification Calibrator Hansenula polymorpha

收稿日期: 2016-01-15 出版日期: 2016-09-25

ZTFLH:

Q816

通讯作者: 李鼎锋 E-mail: lidf@abzymo.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

陈丹, 郭玉争, 班靖洋, 李璐, 王维龙, 李鼎锋, 刘勇. 汉逊酵母重组表达的人胃蛋白酶原Ⅱ校准品研制[J]. 中国生物工程杂志, 2016, 36(9): 38-46.

CHEN Dan, GUO Yu-zheng, BAN Jing-yang, LI Lu, WANG Wei-long, LI Ding-feng, LIU Yong. Development of Recombinant Human PepsinogenⅡ Calibrators Derived from Hansenula polymorpha. China Biotechnology, 2016, 36(9): 38-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160905 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I9/38

[1] Plebani M. Pepsinogens in health and disease. Crit Rev Clin Lab Sci, 1993, 30(3):273-328.
[2] Korstanje A, den Hartog G, Biemond I, et al. The serological gastric biopsy:a non-endoscopical diagnostic approach in management of the dyspeptic patient:significance for primary care based on a survey of the literature. Scand J Gastroenterol, 2002, S236:22-26.
[3] Sipponen P, Härkönen M, Alanko A,et al. Diagnosis of atrophic gastritis from a serum sample. Minerva Gastroenterol Dietol, 2003, 49(1):11-21.
[4] Broutet N, Plebani M, Sakarovitch C, et al. Pepsinogen A, pepsinogen C, and gastrin as markers of atrophic chronic gastritis in European dyspeptics. Br J Cancer, 2003, 88(8):1239-1247.
[5] Foster C, Aktar A, Kopf D, et al. Pepsinogen C:a type 2 cell-specific protease. Am J Physiol Lung Cell Mol Physiol, 2004, 286(2):L382-387.
[6] Kodoi A, Yoshihara M, Sumii K, et al. Serum pepsinogen in screening for gastric cancer. J Gastroenterol, 1995, 30(4):452-460.
[7] Miki K, Morita M, Sasajima M, et al. Usefulness of gastric cancer screening using the serum pepsinogen test method. Am J Gastroenterol, 2003, 98(4):735-739.
[8] Miki K, Urita Y. Using serum pepsinogens wisely in a clinical practice. J Dig Dis, 2007, 8(1):8-14.
[9] Aoki T, Tomaki E, Satoh M, et al. Purification of recombinant human pepsinogens and their application as immunoassay standards. Biochem Mol Biol Int, 1998, 45(2):289-301.
[10] 孟凡红, 金贞姬, 张娟, 等. 重组人血清白蛋白在汉逊酵母中的表达与纯化. 中国生物工程杂志, 2006, 26(12):11-17. Meng F H, Jin Z J, Zhang J, et al. Expression and purification of recombinant human serum albumin in Hansenula polymorpha. China Biotechnology, 2006, 26(12):11-17.
[11] 李鼎锋, 于跃, 霍烛, 等. 用汉逊酵母表达系统产生HPV18 L1蛋白的方法:中国, 201210021524.X.201307-24. Li D F, Yu Y, Huo Z, et al. Method for Producing HPV18 L1 Protein in Hansenula polymorpha. Chinese, 201210021524.X.201307-24.
[12] Kim N, Jung H C.The role of serum pepsinogen in the detection of gastric cancer. Gut Liver, 2010, 4(3):307-319.
[13] Massarrat S, Stolte M. Development of gastric cancer and its prevention. Arch Iran Med, 2014, 17(7):514-520.
[14] Sohn J H, Choi E S, Kim C H,et al.A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1. J Bacteriol, 1996, 178(15):4420-4428.
[15] Saraya R, Gidijala L, Veenhuis M, et al.Tools for genetic engineering of the yeast Hansenula polymorpha. Methods MolBiol, 2014, 1152(1152):43-62.
[16] Quax T E, Claassens N J, Söll D, et al.Codon bias as a means to fine-tune gene expression. Mol Cell, 2015, 59(2):149-161.
[17] Mattanovich D, Branduardi P, Dato L, et al.Recombinant protein production in yeasts. Methods MolBiol, 2012, 824:329-358.

[1]	张玲,曹小丹,杨海旭,李文蕾. 连续流层析技术在亲和层析中的应用及生产放大评估[J]. 中国生物工程杂志, 2021, 41(6): 38-44.
[2]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[3]	蒋丹丹,王云龙,李玉林,张怡青. 含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 22-29.
[4]	谢航航,白红妹,叶超,陈永俊,袁明翠,马雁冰. 易发生聚集的重组HBcAg病毒样颗粒的纯化^*[J]. 中国生物工程杂志, 2020, 40(5): 40-47.
[5]	位薇,常保根,王英,路福平,刘夫锋. Tau蛋白核心片段306~378的异源表达、纯化及聚集特性验证^*[J]. 中国生物工程杂志, 2020, 40(5): 22-29.
[6]	刘珍珍,田大勇. 狂犬病疫苗蔗糖密度梯度离心纯化工艺开发 ^*[J]. 中国生物工程杂志, 2020, 40(4): 25-33.
[7]	朱彤彤,杨磊,刘应保,孙文秀,张修国. 辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 116-123.
[8]	潘炳菊,张宛怡,申会涛,刘婷婷,李中媛,罗学刚,宋亚囝. 甘露寡糖分离纯化研究进展^*[J]. 中国生物工程杂志, 2020, 40(11): 90-95.
[9]	章小毛,郭敬涵,洪解放,陆海燕,丁娟娟,邹少兰,范寰. **UPRE-lac Z为报告基因评价酵母UPR响应初步研究 ^***[J]. 中国生物工程杂志, 2020, 40(10): 1-9.
[10]	胡艳红,龚雪梅,丁柳柳,高嵩,李婷婷. 利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化 ^*[J]. 中国生物工程杂志, 2019, 39(8): 59-65.
[11]	谢玉锋,韩雪梅,路福平. 副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 ^*[J]. 中国生物工程杂志, 2019, 39(5): 72-79.
[12]	付大伟,孙莹莹,徐伟. 融合蛋白NusA-hRI的高效异源表达、纯化及活性分析[J]. 中国生物工程杂志, 2019, 39(3): 21-28.
[13]	景佳美,徐欣,王敏,彭如超,施一. 沙粒病毒聚合酶C端的表达纯化与结晶条件筛选 ^*[J]. 中国生物工程杂志, 2019, 39(12): 18-23.
[14]	朱梦露,王雪雨,刘鑫,路福平,孙登岳,秦慧民. 一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2019, 39(12): 24-34.
[15]	童超迪,吴坚平,杨立荣,徐刚. X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究 ^*[J]. 中国生物工程杂志, 2018, 38(8): 19-25.

Viewed

Full text

Abstract

Cited

Shared

Discussed