中文摘要：

制备高纯度的人ANP32A重组蛋白,并检测其对致癌剂所造成的肝癌细胞损伤的保护作用。运用冷休克表达载体pColdⅡ和无缝克隆技术构建pColdⅡ/ANP32A重组质粒,转化菌诱导表达过夜。菌体超声上清通过镍离子亲和层析柱和钴离子亲和层析柱优化蛋白纯化条件,纯化产物进行Western blot鉴定,分别采用6μg/mL黄曲霉毒素B1（AF-B1）和150 mmol/L乙醇对人肝癌细胞系SMMC7721处理后,加入不同浓度的ANP32A蛋白（0,10,50,100 ng/mL）,24 h后采用CCK-8检测受损伤细胞存活情况,免疫细胞化学法检测PCNA和Ki-67的表达变化。成功获得pColdⅡ/ANP32A原核表达质粒,并实现目的蛋白的可溶性表达,目的蛋白纯度可达到95%。AF-B1和乙醇对SMMC7721细胞具有明显的杀伤作用,CCK-8实验结果提示ANP32A重组蛋白处理组的A值明显高于对照组,其作用具有剂量依赖性。免疫细胞化学法分析结果提示ANP32A可促进损伤细胞中PCNA和Ki-67的表达。ANP32A重组蛋白可在细胞外保护肝细胞,减少致癌剂的损伤作用。

英文摘要：

To prepare highly purified human ANP32A recombination protein,and to observe ANP32A function in extracellular of liver cells when injured by carcinogenic agents, a cold-shock expression vectors pCold II was used. Recombinant plasmid pCold II / ANP32A was constructed by the technology of the seamless cloning. The expression of human ANP32A recombination protein was induced by 0.1 mmol/L IPTG at 16 ℃ overnight in Escherichia coli containing the recombinant plasmid. Then the supernatant lysate of the Escherichia coli was purified by affinity chromatography. The purified target recombinant protein was analyzed by western blot with a specific antibody against human ANP32A. Hepatocellular carcinoma cell line SMMC7721 cells was treated by 6 p,g/mL aria- toxin-B1 （AF-B1） and 150 mmol/L ethanol for 6 hours ,then different concentrations of ANP32A recombination protein （0,10,50, 100 ng/mL） were added. After 24 hours ,viable cells were detected by the CCK-8 analysis, and the color intensity was measured with an ELISA reader at A450. The expression of PCNA and Ki-67 were analyzed by immunocytochemistry, pCold I1/ANP32A prokaryotic expression recombinant plasmid was obtained, and human ANP32A recombinant protein was soluble expressed successfully. The purity of ANP32A recombinant protein was 95% by nickel-ion and cobalt-ion two-step affinity chromatography. AF-B1 and ethanol have obvious damage effect on the SMMC7721 cells. The CCK-8 analysis results suggested that the A450 value of cells in ANP32A protein treatment group was significantly higher than that in the control group,in a dose dependent manner. ANP32A recombinant protein can promote the expression of PCNA and Ki-67 in the damage cells. ANP32A recombinant protein can protect the liver cells from the damage of carcinogens in extracellular.