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目的 构建大鼠肝再生增强因子(rALR)酵母重组表达质粒,在毕赤酵母菌GS115中进行表达。表达产物经超滤方法纯化后在体外进行生物活性研究。方法 以重组质粒pcDNA3.1—rALR为模板,聚合酶链反应(PCR)扩增rALR编码区的DNA,构建重组质粒pPIC9K-rALR,然后电转化至毕赤酵母菌GS115中,在甲醇诱导下进行表达。表达产物经1.5% SDS-PAGE电泳和western blot鉴定后进行超滤纯化。用~3H-TdR掺入法检测重组大鼠ALR(rrALR)体外刺激QGY和HepG2人肝癌细胞株和原代大鼠肝细胞的增殖情况。结果重组质粒经酶切及PCR证实rALR编码区DNA正确插入质粒载体中。重组大鼠ALR被GS115菌以外分泌方式表达,其DNA分子量约为1.5×10~4,与理论预期值相符,western bolt鉴定发现rrALR能与抗人ALR多克隆抗体发生特异性反应,说明人和大鼠ALR抗原性上存在部分交叉。超滤纯化获得大量高纯度的rrALR。在所测定的剂量范围内,rrALR在体外以剂量依赖方式刺激QGY和HepG2肝癌细胞株增殖,但对原代大鼠肝细胞无刺激增殖作用。结论 rrALR在毕赤酵母菌GS115中获得分泌性高效表达,rrALR在体外以剂量依赖方式刺激QGY和HepG2肝癌细胞株增殖,但对原代大鼠肝细胞无刺激增殖作用,表明肝癌细胞和正常肝细胞表达的ALR受体不同。人和大鼠ALR在生物学活性和抗原性上存?
Objective To construct recombinant plasmid of rat liver regeneration augmentation factor (rALR) and express it in Pichia pastoris GS115. The expressed product was purified by ultrafiltration method and its biological activity was studied in vitro. Methods The recombinant plasmid pcDNA3.1-rALR was used as a template to amplify the DNA of rALR coding region by polymerase chain reaction (PCR). The recombinant plasmid pPIC9K-rALR was constructed and then electroporated into Pichia pastoris GS115 under the induction of methanol expression. The expressed product was purified by ultrafiltration using 1.5% SDS-PAGE and western blot. The proliferation of QGY and HepG2 human hepatoma cell lines and primary rat hepatocytes stimulated by recombinant rat ALR (rrALR) in vitro were detected by ~ 3H-TdR incorporation. Results Recombinant plasmids were confirmed by restriction enzyme digestion and PCR. The DNA of rALR coding region was correctly inserted into the plasmid vector. Recombinant rat ALR was secreted by GS115 bacteria and the molecular weight of DNA was about 1.5 × 10 ~ 4, which was consistent with the expected value. Western bolt assay showed that rrALR reacted specifically with anti-human ALR polyclonal antibody, The ALR antigenicity of rats is partly cross-linked. Ultrafiltration purification to obtain a large number of high purity rrALR. In the tested dose range, rrALR stimulated the proliferation of QGY and HepG2 hepatoma cell lines in a dose-dependent manner in vitro without stimulating the proliferation of primary rat hepatocytes. Conclusion rrALR was secreted highly expressed in Pichia pastoris GS115 and rrALR stimulated the proliferation of QGY and HepG2 hepatoma cell lines in a dose-dependent manner in vitro, but it did not stimulate proliferation of primary rat hepatocytes, indicating that hepatoma cells and normal liver Cells express different ALR receptors. Human and rat ALR exist in biological activity and antigenicity?