人甲状旁腺激素在大肠杆菌中的表达

发布时间：2018-08-03 19:34

【摘要】：甲状旁腺激素(PTH)是由甲状旁腺分泌的一种重要的肽类调节激素,在机体的钙磷代谢,骨的形成、分化和发育等相关的诸多环节中起着关键性作用。甲状旁腺激素分子中含有84个氨基酸。近年的研究表明,间歇地小剂量给予PTH可促进骨的合成代谢,因此在治疗骨质疏松方面被认为是最具前景的药物。研究表明PTH(1-34)具有与PTH(1-84)相同的生物功能。据此本研究利用基因工程生产用于治疗骨质疏松症的重组的人甲状旁腺激素(1-34)。利用基因重组技术将人PTH(1-34)基因克隆到表达载体pThioHisA中。重组的表达载体转化到E.coli BL21(DE3)和DH5α中,经异丙醇-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactoside,IPTG)诱导表达。得到融合表达蛋白。经条件优化后,Western-blot分析证实得到了重组人甲状旁腺激素rhPTH(1-34)。经硫酸铵沉淀法纯化后将目的蛋白作用于3T3细胞,结果显示该蛋白可以刺激细胞内cAMP的产生,表明重组的目的蛋白具有一定的生物活性。从以上结果我们可以得出结论:本实验已经利用基因工程技术得到了具有生物活性的重组的人甲状旁腺激素,而且筛选出了一株高效表达菌株。我们证实大肠杆菌BL21(DE3)菌株为理想的表达菌株,从中筛选出的最佳表达条件为:IPTG终浓度为0.5mM,诱导时间为4h。
[Abstract]:Parathyroid hormone (PTH) is an important peptide regulating hormone secreted by parathyroid glands, which plays a key role in the body's calcium and phosphorus metabolism, bone formation, differentiation and development. Parathyroid hormone molecules contain 84 amino acids.
Recent studies have shown that intermittent small doses of PTH can promote bone metabolism and are considered to be the most promising drugs in the treatment of osteoporosis. Research shows that PTH (1-34) has the same biological function as PTH (1-84). Accordingly, the recombinant human parathyroid gland produced by genetic engineering for the treatment of osteoporosis is used in this study. Hormone (1-34).
The recombinant human PTH (1-34) gene was cloned into the expression vector pThioHisA by gene recombination technology. The recombinant expression vector was transformed into E.coli BL21 (DE3) and DH5 alpha, and was induced by isopropanol beta -D- Thioglucoside (Isopropyl- beta -D-thiogalactoside, IPTG). The fusion protein was obtained. After optimization, the Western-blot analysis confirmed the results. The recombinant human parathyroid hormone rhPTH (1-34) was purified by ammonium sulfate precipitation and the target protein was acted on 3T3 cells. The result showed that the protein could stimulate the production of cAMP in the cell, which showed that the recombinant protein had some biological activity.
From the above results, we can draw a conclusion that the recombinant human parathyroid hormone with biological activity has been obtained by genetic engineering, and a highly efficient strain is screened.
We confirmed that Escherichia coli BL21 (DE3) was an ideal expression strain. The optimal expression conditions were as follows: IPTG final concentration was 0.5mM and induction time was 4 h.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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