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目的 :构建人肝再生增强因子原核融合表达载体 ,并对表达产物进行生物活性研究 ,从而为hALR基因修饰的肝细胞移植的细胞来源研究提供实验依据 ,并为人肝再生增强因子的临床应用奠定基础。方法 :以pGEM -T -hALR为模板 ,应用PCR技术扩增出hALRcDNA ,克隆入原核融合表达载体pGEX - 4T - 2 ,限制性内切酶酶切及测序证实序列正确 ;转化大肠杆菌JM10 9:挑取阳性克隆以IPTG诱导表达融合蛋白GST -hALR ,融合蛋白通过谷胱甘肽Sepharose 4B亲和层析纯化后进行凝血酶酶切获得hALR单体 ;采用3 H -thymidine渗入法检测hALR单体的生物学活性。结果 :以 pGEM -T -hALR为模板 ,行PCR扩增后 ,产物于1.5 %琼脂糖凝胶电泳分析 ,可见 380bp特异性条带 ,符合hALRcDNA阅读框架的大小。构建融合蛋白GST-hALR的重组表达质粒pGEX - 4T - 2 -hALR ,经限制性内切酶酶切分析 ,与理论值相符 ,测序证明序列正确 ,片段为正向插入。重组表达质粒转化人肠杆菌JM 10 9。SDS -PAGE电泳分析显示重组菌在约 4 1KD处出现一蛋白条带。纯化、酶切后融合蛋白前体谷胱甘肽S -转移酶约 2 6KD ,hALR约 15KD。薄层扫描蛋白电泳结果表明 ,表达的融合蛋白占细菌可溶性蛋白总量的 31%。纯化hALR加入原代鼠肝细胞、HepG2 细胞培基 ,细胞DNA合成率较对照组
OBJECTIVE: To construct prokaryotic fusion expression vector of human augmenter of liver regeneration and to study the biological activity of the expressed product so as to provide an experimental basis for the study of the origin of cells modified by hALR gene and to lay a foundation for the clinical application of human augmenter of liver regeneration . Methods: The hALR cDNA was amplified by PCR using pGEM-T-hALR as a template, cloned into prokaryotic expression vector pGEX - 4T - 2 and verified by restriction endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli JM109: The positive clones were selected to express the fusion protein GST-hALR induced by IPTG. The fusion protein was purified by glutathione Sepharose 4B affinity chromatography and then cloned into hALR monomers. The hALR monomer was detected by 3 H -thymidine incorporation Biological activity. Results: After PCR amplification with pGEM-T-hALR as template, the product was analyzed by 1.5% agarose gel electrophoresis. The specific band of 380bp was observed, which accorded with the size of hALR cDNA. The recombinant plasmid pGEX - 4T - 2 - hALR was constructed by restriction endonuclease digestion analysis, which was consistent with the theoretical value. Sequencing proved that the sequence was correct and the fragment was inserted in the forward direction. Recombinant expression plasmid was transformed into E. coli JM109. SDS-PAGE electrophoresis analysis showed that the recombinant bacteria appeared at about 4 1KD a protein band. After purification, the fusion protein precursor glutathione S - transferase about 26KD, hALR about 15KD. The results of TLC scanning indicated that the expressed fusion protein accounted for 31% of the total bacterial soluble protein. Purified hALR was added into primary murine hepatocytes and HepG2 cells cultured in vitro. The DNA synthesis rate of cells was higher than that of control group