谷氨酰胺:6-磷酸果糖酰胺基转移酶多克隆抗体的制备及应用

Preparation and Application of the Polyclonal Antibody Against Glutamine:Fructose 6-Phosphate Amidotransferase

目的:采用原核表达系统表达和纯化重组谷氨酰胺∶6-磷酸果糖酰胺基转移酶(GFAT),制备GFAT多克隆抗体,并用以研究在糖尿病发生发展过程中GFAT的表达情况。方法:通过生物信息学分析其抗原性和属间同源性,选择需要的基因区域;利用RT-PCR扩增小鼠肝脏cDNA中GFAT基因片段,克隆到表达载体pET28b中;在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组GFAT;用纯化的重组GFAT免疫BALB/c小鼠后得到多克隆抗体;用Western印迹检测正常小鼠、高血糖小鼠及胰岛素抵抗小鼠的组织GFAT的表达。结果:Western印迹分析表明,制备的GFAT抗体具有较高的特异性,可特异性识别重组GFAT和正常小鼠肝脏、肾脏、骨骼肌和肺组织的GFAT;与正常小鼠相比,高脂饲料诱导的胰岛素抵抗小鼠肌肉组织的GFAT表达升高约1.8倍,肝脏组织则略有升高;高血糖小鼠肌肉组织和肝脏组织的GFAT表达也略有上升,但无统计学差异。结论:利用原核表达及Ni-NTA纯化系统可制备小鼠GFAT多克隆抗体;正常小鼠骨骼肌GFAT表达较高;肌肉组织的GFAT表达在胰岛素抵抗小鼠显著性升高,而与小鼠血糖水平无明显相关性。

Objective： To further study the function of glutamine：fructose 6-phosphate amidotransferase（GFAT）, the polyclonal antibody against GFAT were prepared. Methods： The antigenic of GFAT was analyzed and the half length of GFA T gene was cloned to the pET-28b plasmid. Then the recombinant plasmid was transformed into Escherichia coli BL21（DE3） and the expressed recombinant protein was purified by Ni-NTA column. The purified recombinant protein was injected to generate the polyclonal antibody. The expression of GFAT in muscle and liver tissue of alloxan-induced hyperglycaemia mice and high fat diet-induced insulin resistance mice were analyzed by Western blot. Results： Based on the experiments, the polyclonal antibody was obtained. The Western blot results showed that the antibody could recognize GFAT from mouse tissues specifically. Compared with normal mice, the expression of GFAT in skeletal muscle of high fat diet-in- duced insulin resistance mice was significantly increased by 1.8 fold; the expression of GFAT in liver tissue was upgraded slightly. The expression of GFAT in muscle and liver tissue of alloxan-induced hyperglycaemia mice were increased slight- ly, too. Conclusion： The polyclonal antibody against GFAT was prepared successfully, which supplied a good tool for further studying on the relationship during GFAT, insulin resistance and diabetes. The expression of GFAT in skeletal muscle was much higher than that of other tissues. GFAT in skeletal muscle of high fat diet-induced insulin resistance mice was significantly increased, and had no positive relationship with the level of serum glucose.