微囊化和基因修饰的肝细胞移植--人肝再生增强因子(hALR)的原核表达、纯化及生物活性研究

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JM109：桃取阳性克隆以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^3H-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JM109。SDS－PAGE电泳分析显示重组菌在约41KD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26KD，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合成率较对照组均有显著提高（P＜0．01）。结论：成功构建融合蛋白GST－hALR的重组表达质粒pGEX-4T-2-hALR，限制性内切酶酶切及序列测定证明载体插入正确，并成功转化大肠杆菌JM109得阳性克隆。采用含融合蛋白GST的原核表达载体pGEX-4T-2表达hALR，其突出优点在于表达产率高；重组蛋白主要以可溶形式存在于胞质中，融合蛋白的分离纯化筛单易行。纯化分离后所得hALR能刺激体外培养的原代鼠肝细胞、HepG2细胞DNA合成，具有生物活性。

Transplantation of the Encapsulated and Gene Modified Hepatocyte --Transplantation of the Encapsulated and Gene Modified Hepatocyte of human Augmenter of Liver Regeneration and the Activity of its Product

Objective:To construct the Prokaryotic expression vector of GST- hALR fusion protein, and evaluate the biological activity of the expressive product. So it provides experimental evidence for both the study of hepatocellular resource of hALR gene modified hepatocyte transplantation and the clinical application of hALR.Methods: By adopting PCR technology, the hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and the cDNA was subcloned into the Prokaryotic expression vector pGEX-4T-2. The recombined vector, pGEX-4T-2-hALR, was identified by enzyme digestion and DNA sequencing and transformed into E.coli.JM109. The positive clone selected was induced by the expression of GST- hALR fusion protein by IPTG, then the fusion protein was purified by glutathione S-transferase(GST) Sepharose 4B affinity chromatography,cleaved by thrombin and the hALR monomer was obtained. Alter that, the activity of the hALR monomer was detected by measuring 3 Hthymidine incorporation.Results:The produce of PCR from plasmid pGEM-T-hALR was examined by 1.5% agarose gel electrophoresis, the specific strap was coincided with hALRcDNA. The recombined expression vector pGEX-4T-2-hALR digested by enzyme was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction.The recombined vector was transformed into E.coli.JM109.SDS-PAGE proved that expressive fusion protein induced showed a single band with a molecular weight of 41KD. The product was purified and cleaved. The molecular weights of GST and hALR were 26KD, 15KD respectively. The recombinant fusion protein accounted for 31% of total soluble protein of bacterial lysate by scanning.HALR added to the culture medium of adult rat hepatocyte in primary culture and HepG2 cell line can significantly enhance the rate of DNA synthesis compared with the relevant control groups( P <0.01).Conclusion: Fusion expression vector pGEX-4T-2-hALR was reconstructed successfully. Inserting vectors was proved to be accurate by the methods of the enzyme digestion and sequencing.Transforming E.coli.JM109 and obtaining the positive clone were successfully performed.Expressing hALR by vector pGEX-4T-2 is highly efficient which makes it a prominent virtue. The recombinant GST-hALR expressed in cytoplasma was soluble. Therefore all tile procedures of purification and cleavage of fusion protein to release free hALR were easy and simple.The purified hALR have the ability of stimulating DNA synthesis of adult rat hepatocyte in primary culture and HepG 2 cell in vitro.