Expression, purification and bioactivity of human augmenter of liver regeneration

AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

瞄准：为肝新生(hALR ) 的原核生物、真核细胞的人的 augmenter 构造表示向量并且学习他们的生物活动。方法：hALRcDNA 克隆从原生质标志 pGEM-T-hALR 被获得，并且 cDNA 是克隆进 prokatyotic 表示向量 pGEX-4T-2 的潜水艇。recombinant 向量和 pGEX-4T-2hALR 被酶消化并且 DNA 定序识别并且转变了成 E 关口 i JM109。断然选择的克隆被 GST-hALR 熔化蛋白质的表示与 IPTG 导致，然后，熔化蛋白质被 glutathine s-transferase (GST ) 净化 sepharose 4B 亲密关系层析，由测量 H 胸腺嘧啶核甙加入由凝血酵素劈开， hALR 单体被获得并且检测。结果：从原生质标志 pGEM-T-hALR 的 PCR 的产品被 1.5% sepharose 电气泳动检验。特定的带与理论的是重合的。顺序是精确的，酶消化的 pGEX-4T-hALP 与理论的是重合的。顺序是精确的，碎片在积极方向被插入。recombinant 向量被转变成 E 关口 i JM109。SDS 页证明导致的富有表达力的熔化蛋白质与 41 kDa 的分子量给一个单身的乐队看了。产品被净化并且劈开。GST 和 hALR 的分子量是 26 kDa， 15 kDa 分别地。recombinant 熔化蛋白质说明了细菌的溶解产物的 31% 全部的可溶的蛋白质。在主要文化和 HepG2 房间线加到成年老鼠 hepatocytes 的培养基的 HALR 能显著地与相关控制组相比提高 DNA 合成的率(P < 0.01 ) 。结论：净化的 hALR 有能力刺激 DNA 在主要文化和 HepG2 房间在试管内的成年老鼠 hepatocytes 的合成，和罐头为它的临床的申请提供证据。