摘要
目的:观察可溶性表达的融合蛋白GST-hRI对CCl4引起的急性肝损伤的保护作用。方法:构建pGEX-6p-1-hri表达质粒。在0.5 mol/L IPTG浓度下,调整温度诱导目的融合蛋白的最大可溶性表达。用Western Blotting检测GST-hRI的表达,用亲和层析法对GST-hRI进行分离纯化。将纯化后的GST-hRI腹腔注射给BALB/c小鼠,预先保护7 d后,再腹腔注射CCl4造成急性肝损伤。检测小鼠血清谷丙转氨酶、谷草转氨酶水平和肝脏的超氧化物歧化酶、黄嘌呤氧化酶、丙二醛、谷胱甘肽、谷胱甘肽过氧物酶指标,并在光镜下检测肝细胞的损伤程度。结果:0.5 mmol/L IPTG,26℃诱导表达8 h,超声破碎菌体所得到的上清中可溶性GST-hRI的表达量约占超声离心后上清总蛋白4.5%,与沉淀中的比例约为27%。与CCl4损伤组相比,GST-hRI治疗组的谷丙转氨酶、谷草转氨酶和丙二醛水平明显下降(P<0.05),谷胱甘肽过氧化物酶、超氧化物歧化酶和谷胱甘肽水平明显增加(P<0.05),黄嘌呤氧化酶水平基本没有变化。与hRI治疗组相比,GST-hRI治疗组的各项指标差异有统计学意义(P<0.05)。与维生素C处理组相比,GST-hRI处理组的各项指标差异无统计学意义(P>0.05)。结论:可溶性表达的融合蛋白GST-hRI具有对抗CCl4对小鼠肝损伤的作用,这为hRI的应用提供了实验基础。
Objective To observe the protective effect of the resoluble fusion protein GST-hRI expressed in the E.coli BL21 transfected by pGEX-6P-1-hri on the CCl4 damaged mice liver.Methods The pGEX-6P-1-hri was constructed by inserting GST at the upstream of hRI cDNA,and the expression of fusion GST-hRI was induced at the concentration of 0.5 mol/L IPTG under the conditions of several temperatures.The expression of GST-hRI was detected by Western blotting and it was purified by affinity chromatography.BALB/c mice treated with peritoneal injection of purified GST-hRI. After 7 days, CCl4 was injected peritoneally to cause the acute hepatic damage of mice. The serum levelsofALT, AST, the liver cytosol levels of SOD,XOD,GSH,MDA and GSH-Px were deteced. The damaged hepatocytes were observed under microscope. Results The GST-hRI content in the supernatant of disintegrated thallus accounted for 4.5% in the total protein. The levels of ALT, AST and MDA in GST-hRI group were significantly lower（P〈0.05）, GSH-Px, SOD and GSH were remarkably higher（P〈0.05）than those of CCl4 treated group. The XOD levels did not change. Conclusion The GST-hRI of resoluble expression has the protective effect on the acute hepatic damage caused by CCl4 in mice, which establishs a certain experimental basis to develop hRI application in the future.
出处
《中华实用诊断与治疗杂志》
2009年第8期784-787,共4页
Journal of Chinese Practical Diagnosis and Therapy
