【摘要】：甲狀旁腺素相關(guān)蛋白(parathyroid hormone-related protein,PTHrP)最初作為一種引起惡性腫瘤患者發(fā)生高鈣血癥的蛋白被發(fā)現(xiàn)。隨著對其研究的深入,證實其在大多數(shù)組織器官都有所表達(dá),提示其在正常細(xì)胞生長分化中的重要性。PTHrP主要通過自分泌、旁分泌和胞內(nèi)分泌的作用方式發(fā)揮作用。PTHrP被蛋白酶作用后,產(chǎn)生一組具有不同生物活性的多肽,它的作用靶組織與靶器官涉及非常廣泛。PTHrP的這些生理功能,預(yù)示著PTHrP未來在臨床上廣泛的應(yīng)用前景。 本實驗采用TKM法自人血細(xì)胞中提取基因組DNA,并以之為模板PCR擴(kuò)增hPTHrP1-141編碼基因。將該基因克隆入pGEM-T easy載體,并進(jìn)行DNA序列分析。在此基礎(chǔ)上,將編碼hPTHrP1-141和hPTHrP1-86的基因片段分別克隆到表達(dá)載體pQE-30Xa上,并轉(zhuǎn)化入E. coli M15[pREP4],進(jìn)行融合表達(dá)。表達(dá)條件經(jīng)優(yōu)化后,rhPTHrP1-141和rhPTHrP1-86的表達(dá)量分別達(dá)到60mg/L培養(yǎng)基和4mg/L培養(yǎng)基。重組蛋白rhPTHrP1-141和rhPTHrP1-86分別占菌體可溶性蛋白的34.64%和6.38%。目的蛋白含6×His純化標(biāo)簽,可以經(jīng)金屬螯合親和層析吸附于Ni-NTA樹脂層析柱,梯度增加沖洗液中咪唑濃度以除去非特異吸附的雜蛋白后,將目的蛋白洗脫,超濾離心法濃縮。體外培養(yǎng)大鼠骨肉瘤細(xì)胞系UMR 106,以rhPTH1-34標(biāo)準(zhǔn)品作為陽性對照,將PTHrP1-141和PTHrP1-86加入細(xì)胞培養(yǎng)液,使其終濃度分別為10~(10)mol/L、10~(-9)mol/L、10~(-8)mol/L、10~(-7)mol/L、10~(-6)mol/L和10~(-5)mol/L。PTHrP能與存在于UMR 106細(xì)胞膜上的與G蛋白相偶連的PTH1型受體(PTH1R)結(jié)合,激活
[Abstract]:Parathyroid hormone-associated protein (parathyroid hormone-related protein,PTHrP) was originally identified as a protein that causes hypercalcemia in patients with malignant tumors. With the further study of PTHrP, it has been confirmed that it is expressed in most tissues and organs, suggesting its importance in normal cell growth and differentiation. PTHrP is mainly expressed through autocrine. Paracrine and cytosolic endocrine function. PTHrP is treated by protease to produce a group of polypeptides with different biological activities. Its target tissues and target organs are involved in a wide range of physiological functions of PTHrP. It indicates that PTHrP will be widely used in clinic in the future. In this study, genomic DNA, was extracted from human blood cells by TKM method and used as template PCR to amplify hPTHrP1-141 coding gene. The gene was cloned into pGEM-T easy vector and sequenced by DNA. On this basis, the gene fragments encoding hPTHrP1-141 and hPTHrP1-86 were cloned into the expression vector pQE-30Xa, and transformed into E. coli M15 [pREP4] for fusion expression. After optimized expression conditions, the expression of rhPTHrP1-141 and rhPTHrP1-86 reached 60mg/L medium and 4mg/L medium respectively. The recombinant protein rhPTHrP1-141 and rhPTHrP1-86 accounted for 34.64% and 6.38% of the soluble protein, respectively. Objective the protein contains 6 脳 His purified label and can be adsorbed on Ni-NTA resin column by metal chelating affinity chromatography. The target protein is eluted and concentrated by ultrafiltration centrifugation by gradient increasing the concentration of imidazole in the flushing solution to remove the non-specific adsorbed impurity. Rat osteosarcoma cell line UMR 106 was cultured in vitro. PTHrP1-141 and PTHrP1-86 were added into cell culture medium with rhPTH1-34 standard as positive control. The final concentration of PTHrP1-141 and PTHrP1-86 were 10 ~ (10) mol/L,10~ (-9) mol/L, respectively. 10 ~ (-8) mol/L,10~ (-7) mol/L,10~ (-6) mol/L and 10 ~ (-5) mol/L.PTHrP can bind and activate PTH1 type receptor (PTH1R) which is coupled to G protein on UMR 106 cell membrane.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q51

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前4條

1 張明新;HLA-B寡核苷酸分型芯片制備方法的建立及初步應(yīng)用[D];天津醫(yī)科大學(xué);2006年

2 馬香書;大腸桿菌偏嗜性人促甲狀腺激素（hTSH）β亞基編碼DNA序列的克隆與表達(dá)[D];天津醫(yī)科大學(xué);2007年

3 張瑞;HLA寡核苷酸分型芯片的制備及其初步應(yīng)用[D];天津醫(yī)科大學(xué);2007年

4 王春雨;大鼠DJ-1基因的克隆和表達(dá)[D];天津醫(yī)科大學(xué);2008年

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本文編號：2321577