【摘要】：甲狀旁腺激素(PTH)是由甲狀旁腺分泌的一種重要的肽類調(diào)節(jié)激素,在機(jī)體的鈣磷代謝,骨的形成、分化和發(fā)育等相關(guān)的諸多環(huán)節(jié)中起著關(guān)鍵性作用。甲狀旁腺激素分子中含有84個氨基酸。 近年的研究表明,間歇地小劑量給予PTH可促進(jìn)骨的合成代謝,因此在治療骨質(zhì)疏松方面被認(rèn)為是最具前景的藥物。研究表明PTH(1-34)具有與PTH(1-84)相同的生物功能。據(jù)此本研究利用基因工程生產(chǎn)用于治療骨質(zhì)疏松癥的重組的人甲狀旁腺激素(1-34)。 利用基因重組技術(shù)將人PTH(1-34)基因克隆到表達(dá)載體pThioHisA中。重組的表達(dá)載體轉(zhuǎn)化到E.coli BL21(DE3)和DH5α中,經(jīng)異丙醇-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactoside,IPTG)誘導(dǎo)表達(dá)。得到融合表達(dá)蛋白。經(jīng)條件優(yōu)化后,Western-blot分析證實(shí)得到了重組人甲狀旁腺激素rhPTH(1-34)。經(jīng)硫酸銨沉淀法純化后將目的蛋白作用于3T3細(xì)胞,結(jié)果顯示該蛋白可以刺激細(xì)胞內(nèi)cAMP的產(chǎn)生,表明重組的目的蛋白具有一定的生物活性。 從以上結(jié)果我們可以得出結(jié)論:本實(shí)驗(yàn)已經(jīng)利用基因工程技術(shù)得到了具有生物活性的重組的人甲狀旁腺激素,而且篩選出了一株高效表達(dá)菌株。 我們證實(shí)大腸桿菌BL21(DE3)菌株為理想的表達(dá)菌株,從中篩選出的最佳表達(dá)條件為:IPTG終濃度為0.5mM,誘導(dǎo)時間為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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346

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