中文摘要：

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pSumo-Mut表达载体中，构建小泛素相关修饰物-骨钙素质粒（pSumo-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pSumo-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，16．7mmol·L^-1葡萄糖下，胰岛素分泌为（37．64±3．80）μu·L^-1，16．7mmol·L^-1葡萄糖＋0．03ng·mL^-1。Sumo-BGLAP融合蛋白作用时，胰岛素分泌为（63．91±4．67）μU·L^-1，16．7mmol·L^-1葡萄糖＋0．3ng·mL^-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。

英文摘要：

Objective To further study the regulatory mechanisms of un- carboxylated osteocalcin on the glucose metabolism, rat uncarboxylated osteocalcin fusion protein was expressed, purified and identified. Methods The rat osteocalcin gene （BGLAP） was cloned into pSumo- Mut expression vector. The pSumo -BGLAP plasmid was ob- tained and identified. Through transformed in prokaryotic host bacterium and induced by isopropyl β - D - thiogalactoside （IPTG） and 11 ℃ low temperature, the Sumo - BGLAP fusion protein was expressed. The purification and activity identification of the fusion protein was performed by Ni^2＋ affinity chromatography and insulin secretion experiments, re- spectively. Results The enzyme digestion and sequencing results of the plasmid showed that the recombinant plasmid pSumo - BGLAP was con- structed successfully, which matched the target gene and protein. After the transformation, induction and purification, the high purity Sumo- BGLAP fusion protein was obtained. The data from insulin secretion experiments show that the fusion protein can promote insulin secretion under 16.7 mmol · L^-1 glucose conditions [ （37.64 ± 3.80）μU·L^-l at 16.7 mmol · L^-l glucose, （63.91±4.67） μU · L^-1 at 16.7 mmol · L^-1 glucose ＋0.03 ng · mL^-l Sumo-BGLAP, （68.47 ± 5.83） μU · L^-1 at 16.7 mmol · L^-1 glucose ＋0.3 ng · mL^-1 Sumo - BGLAP, P 〈0. 01 ） 1- Conclusion The rat uncarboxylated osteocalcin fusion protein is successfully achieved and purified. The fusion protein with biological activity can be used to further study the regulatory mechanisms of uncarboxylated osteocalcin on the glucose metabolism.