| 白脂素包涵体表达纯化及其功能评价 |
| |
| 引用本文： | 韦雪静,敖青青,孟玲,徐怡璐,陆彩玲,唐深,王新航,李习艺. 白脂素包涵体表达纯化及其功能评价[J]. 南方医科大学学报, 2020, 40(1): 67-72. DOI: 10.12122/j.issn.1673-4254.2020.01.10 |
| |
| 作者姓名： | 韦雪静 敖青青 孟玲 徐怡璐 陆彩玲 唐深 王新航 李习艺 |
| |
| 摘 要： | 目的 构建白脂素的原核表达质粒,表达及纯化重组白脂素蛋白,并初步探索其功能,为进一步研究白脂素作用机制及其生理病理作用提供依据。方法 利用基因工程技术构建表达白脂素BL21菌株,经异丙基硫代半乳糖苷诱导,梯度尿素复性,亲和层析纯化及去内毒素处理得到可用于细胞和动物实验的重组白脂素。采用血糖仪检测重组白脂素腹腔注射后C57小鼠血糖变化情况。Alamar Blue检测重组白脂素作用24 h对MIHA细胞活性的影响;重组白脂素刺激MIHA细胞24 h,用蒽酮法检测糖原含量。结果 在37 ℃,A600 nm=0.8,IPTG终浓度为1 mmol/L诱导4 h目的蛋白表达效果较好。通过纯化和去内毒素处理,重组白脂素纯度大于95%,内毒素含量小于1 EU/mg,可以用于动物与细胞实验。腹腔注射重组白脂素60 min后C57小鼠血糖升高至峰值（重组白脂素组 vs 对照组P=0.021）,120 min恢复到正常水平（重组白脂素组vs 对照组P=0.03）。重组白脂素刺激MIHA细胞24 h,对细胞活性没有影响;检测糖原发现,不同浓度白脂素组糖原与对照组比较差异有统计学意义（P1=0.013,P2=0.036, P3=0.011）。结论 通过原核表达系统成功表达及纯化了白脂素蛋白,该方法纯化后的白脂素具有降低MIHA细胞糖原含量及升高小鼠血糖的作用,这为白脂素功能的进一步研究提供了理论依据。
|
Expression,purification and functional assessment of asprosin inclusion body |
| |
| Abstract: | Objective The obtain purified recombinant asprosin and test its functions. Methods The recombinant plasmid ofpET-22b-asprosin was constructed and transformed into competent E.coli BL (DE3) strain. After IPTG-induced expression,asprosin inclusion body was renatured by gradient urea and purified by Ni-NTA affinity chromatography column followed byremoval of endotoxin to obtain recombinant asprosin for use in cells and animals experiments. C57 mice were injectedintraperitoneally with the recombinant asprosin and blood glucose was detected using a blood glucose meter. Alamar Blueassay was used to evaluate of the effect of the recombinant asprosin on the viability of MIHA cells, and cellular glycogencontent was detected using the anthrone method. Results At the absorbance at 600 nm of 0.8, induction of the recombinanthost bacteria with 1 mmol/L IPTG at 37 ℃ for 4 h optimally induced the expression of asprosin inclusion body. Afterpurification and endotoxin removal, the purity of the recombinant asprosin exceeded 95% with the content of endotoxin below1 EU/mg. In C57 mice, intraperitoneal injection with recombinant asprosin significantly increased blood glucose level, whichreached the peak level at 60 min following the injection (P=0.021) and recovered the normal level at 120 min (P=0.03).Treatment with the recombinant asprosin for 24 h did not cause obvious adverse effect on the viability of MIHA cells butsignificantly lowered glycogen content in the cells (P<0.05). Conclusion We successfully obtained recombinant asprosin usinga prokaryotic expression system. The recombinant asprosin can decrease glycogen content in MIHA cells and increase bloodglucose level in mice. |
| |
| Keywords: | |
|
| 点击此处可从《南方医科大学学报》浏览原始摘要信息 |
|
点击此处可从《南方医科大学学报》下载免费的PDF全文 |
|
