大鼠未羧化骨钙素融合蛋白的表达纯化及活性鉴定

发布时间：2018-01-22 22:58

本文关键词： 小泛素相关修饰物骨钙素原核表达胰岛素分泌　出处：《中国临床药理学杂志》2017年09期 　论文类型：期刊论文

【摘要】：目的研究表达和纯化大鼠未羧化骨钙素融合蛋白并鉴定其活性。方法将大鼠的骨钙素(BGLAP)基因克隆到p Sumo-Mut表达载体中,构建小泛素相关修饰物-骨钙素质粒(p Sumo-BGLAP)并进行质粒酶切及测序鉴定。质粒在大肠杆菌的表达系统中转化后,利用异丙基-β-D-硫代半乳糖苷(IPTG),11℃低温诱导方法,表达出小泛素相关修饰物-骨钙素(Sumo-BGLAP)融合蛋白。融合蛋白通过Ni亲和纯化方法被纯化,通过胰岛素分泌实验被鉴定生物活性。结果质粒的酶切及测序结果显示与目的基因的序列和蛋白分子量吻合,表明质粒p Sumo-BGLAP构建成功。经转化、诱导和纯化后较高纯度的Sumo-BGLAP融合蛋白被获得。胰岛素分泌实验结果显示,16.7 mmol·L~(-1)葡萄糖下,胰岛素分泌为(37.64±3.80)μU·L~(-1),16.7 mmol·L~(-1)葡萄糖+0.03 ng·m L~(-1)Sumo-BGLAP融合蛋白作用时,胰岛素分泌为(63.91±4.67)μU·L~(-1),16.7 mmol·L~(-1)葡萄糖+0.3 ng·m L~(-1)Sumo-BGLAP融合蛋白作用时,胰岛素分泌为(68.47±5.83)μU·L~(-1)(均P0.01)。结论大鼠未羧化骨钙素的融合蛋白被成功表达和纯化,并且有生物活性,能用于进一步研究其对葡萄糖代谢的调节作用。
[Abstract]:Objective to study the expression and purification of rat uncarboxylated osteocalcin fusion protein and identification of its activity. Methods the rat osteocalcin (BGLAP) gene was cloned into P Sumo-Mut expression vector, construction of small ubiquitin related modifier (P Sumo-BGLAP) - osteocalcin plasmid and plasmid enzyme digestion and sequencing. The plasmid was transformed into expression system in Escherichia coli, using isopropyl thiogalactoside beta -D- (IPTG), the low temperature of 11 DEG C for inducing expression of small ubiquitin related modifier - Osteocalcin (Sumo-BGLAP) fusion protein. The fusion protein by Ni affinity purification method was pure, secretion was identified by insulin. The biological activity of plasmid digestion and sequencing results showed that the molecular weight of the protein sequence and the target gene with that the plasmid P Sumo-BGLAP was constructed successfully. After transformation, after induction and purification of Sumo-BGLAP fusion protein with high purity was obtained. Islet Hormone secretion, the experimental results show that the 16.7 mmol - L~ (-1) glucose, insulin secretion (37.64 + 3.80) U - L~ (-1), 16.7 mmol - L~ (-1) ng - M L~ glucose +0.03 (-1) Sumo-BGLAP fusion protein, insulin secretion is (63.91 + 4.67) U L~ (-1), 16.7 mmol - L~ (-1) ng - M L~ glucose +0.3 (-1) Sumo-BGLAP fusion protein, insulin secretion is (68.47 + 5.83) U - L~ (-1) (P0.01). Conclusion the rat uncarboxylated osteocalcin fusion protein was successfully expressed and purified. And there is a biological activity, can be used to further study the regulatory effects on glucose metabolism.

【作者单位】：山西医科大学药理教研室;山西医科大学细胞生理学省部共建教育部重点实验室;山西医科大学儿科医学系;山西医科大学第一医院内分泌科;
【基金】：国家自然科学基金资助项目(81273564,81373464,81670710) 山西省留学回国人员科技活动项目择优资助基金资助项目(2016-97)
【分类号】：R3411
【正文快照】： (1.山西医科大学,a.药理教研室;b.细胞生理学省部共建教育部重点实验室;c.儿科医学系,太原030001;2.山西医科大学第一医院内分泌科,太原030001)GAO Jing-ying1a,1b,1c,REN Le-le1a,DING Ya-qin1a,ZHONG Xiang-qin1a,BAI Tao1a,1b,2,LIU Yun-feng2,ZHANG Yi1a,1b(1.a.Departmen

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