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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P<0.01)。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。
Objective To study the expression and purification of non-carboxylated osteocalcin fusion protein in rats and to identify its activity. Methods The rat osteocalcin (BGLAP) gene was cloned into p Sumo-Mut expression vector to construct p Sumo-BGLAP, a small ubiquitin-related modification. The plasmid was digested with plasmid and sequenced. Plasmids were transformed into E.coli expression system and then expressed in small amount of ubiquitin-related osteocalcin (COOH) by using low temperature induction method at 11 ℃ with isopropyl-β-D-thiogalactopyranoside (IPTG) BGLAP) fusion protein. The fusion protein was purified by Ni affinity purification and bioactivity was identified by insulin secretion assay. Results Plasmid digestion and sequencing results showed that the sequence and protein molecular weight of the target gene were consistent, which indicated that the plasmid pStoo-BGLAP was successfully constructed. After transformation, induction and purification of higher purity Sumo-BGLAP fusion protein was obtained. Insulin secretion assay showed that insulin secretion was (37.64 ± 3.80) μU · L -1, 16.7 mmol·L -1 glucose + 0.03 ng · m -1 under 16.7 mmol·L -1 glucose, The insulin secretion was (63.91 ± 4.67) μU · L -1, 16.7 mmol·L -1 glucose + 0.3 ng · m L ~ (-1) in the L -1 (-1) Sumo-BGLAP fusion protein, 1) The insulin secretion was (68.47 ± 5.83) μU · L -1 for Sumo-BGLAP fusion protein (all P <0.01). Conclusion The fusion protein of osteocalcin is successfully expressed and purified in rat and has biological activity. It can be used to further study its regulation on glucose metabolism.