刘晓磊;张慧丽;王俊山;韩静;常丽青;鲁琨;王战争;
LIU Xiao-lei;ZHANG Hui-li;WANG Jun-shan;HAN Jing;CHANG Li-qing;LU Kun;WANG Zhan-zheng;Zhengzhou Biocell Biotechnology Co., Ltd.;

• 1:郑州人福博赛生物有限责任公司
• 2:郑州市第七人民医院
• 3:河南康鑫生物技术有限公司

摘要(Abstract)：

目的表达并纯化重组人催乳素(prolactin,PRL)蛋白,并检测其抗原性及稳定性。方法以商品化的PRL c DNA为模板,PCR扩增目的基因,定向克隆至质粒p ET25中,构建重组原核表达质粒PRL-p ET25b,转化大肠埃希菌后诱导表达,产物经Ni-NTA亲和层析柱纯化,SDS-PAGE鉴定后,采用Bradford法测定蛋白浓度,PRL定量试剂盒分析抗原性,置-20、4和37℃9 d后,分析稳定性。结果重组表达质粒经双酶切及测序鉴定证明构建正确,表达的重组PRL蛋白相对分子质量约30 000,以包涵体形式存在,表达量占菌体总蛋白的15%,纯度大于90%,总蛋白浓度为0.796 mg/ml。重组PRL蛋白于-20、4和37℃放置9 d,浓度变化较小,偏差在10%以内。结论获得了高纯度的重组PRL融合蛋白,抗原性及稳定性均较好,可应用于试剂盒标准品或校准品的制备。
Objective To express and purify recombinant human prolactin(PRL) protein and determine its antigenicity and stability. Methods Target gene was amplified by PCR using commercial PRL c DNA as a template and cloned into plasmid p ET25 b. The constructed recombinant plasmid PRL-p ET25 b was transformed to E. coli and induced with IPTG,and the expressed protein was purified by Ni-NTA chromatography, identified by SDS-PAGE, determined for concentration by Bradford method, and analyzed for antigenicity by PRL quantitative detection kit, then stored at-20, 4 an 37 ℃ for 9 d respectively and evaluated for stability. Results Restriction analysis and sequencing proved that recombinant plasmid PRL-p ET25 b was constructed correctly. The expressed PRL protein, with a relative molecular mass of about 30 000, exist-ed in a form of inclusion body and contained about 15% of total somatic protein, of which the purity was more than 90%and the total protein content was 0. 796 mg / ml. After storage at-20, 4 an 37 ℃ for 9 d, the variation of PRL concentration was less than 10%. Conclusion Highly purified recombinant PRL fusion protein was obtained, which showed good antigenicity and stability and might be used for the preparation of standard kit and calibrator.

关键词(KeyWords)： PRL;原核细胞;基因表达;纯化;抗原性;稳定性
PRL;Prokaryotic expression;Gene expression;Purification;Antigenicity;Stability

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 刘晓磊;张慧丽;王俊山;韩静;常丽青;鲁琨;王战争;
LIU Xiao-lei;ZHANG Hui-li;WANG Jun-shan;HAN Jing;CHANG Li-qing;LU Kun;WANG Zhan-zheng;Zhengzhou Biocell Biotechnology Co., Ltd.;

DOI: 10.13200/j.cnki.cjb.000845

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