人重组催乳素蛋白的原核表达、纯化及其稳定性分析

Prokaryotic expression,purification and stability of recombinant human prolactin protein

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 antigenieity and stability. Methods Target gene was amplified by PCR using commercial PRL eDNA as a template and cloned into plasmid pET25b. The constructed recombinant plasmid PRL-pET25b 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-pET25b 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.