鸡骨钙素蛋白的原核表达及其生物学活性

Expression of chicken osteocalcin in Escherichia coli and its biologic activity

为获得具有生物活性的骨钙素蛋白,以鸡颅骨提取的总RNA为模板,RT-PCR法克隆鸡骨钙素成熟肽DNA,连接至原核表达载体pET-32a(+),构建表达质粒pET-32a(+)-rchOC。质粒转化Rosetta-gami(DE3)pLyS宿主菌,IPTG诱导表达含His标签的重组蛋白His-rchOC。肠激酶酶切去除融合蛋白中的His标签后获得重组骨钙素蛋白(rchOC)。通过与羟基磷灰石结合试验、MTT及PNPP法分析该蛋白生物学活性。结果显示:重组骨钙素蛋白能与羟基磷灰石结合,抑制成骨细胞中碱性磷酸酶的表达,但不影响成骨细胞的增殖。结论:经原核表达系统已成功获得鸡重组骨钙素蛋白,且该蛋白具有生物活性。

The aim of this study is to obtain the reeombinant chicken osteocalcin protein（rehOC） with biologic activity by Escherichia coll. Chicken osteocalcin gene was cloned from cranial bone of chick embryo and ligated into the multiple chining sites of pET-32a （＋） for the construction of pET-32a（＋）-rchOC. The protein,rchOC with His-tag（ His-rchOC ）was expressed in E. coli Rosetta-gami （DE3） pLyS under IPTG induction and was digested with recombinant enterokinase to obtain rehOC without His-tag. The biological activities of rchOC were analyzed using methods of hydroxyapatite binding,MTT and PNPP. The results showed that rchOC can bind hydroxyapatite and inhibit alkaline phosphatase activity of osteoblast,but did not affect the proliferation of osteohlast. In conclusion, the active recombinant chicken osteocalcin protein was prepared successfully in this experiment.