本文關(guān)鍵詞： 人增殖誘導(dǎo)配體(APRIL) 腫瘤壞死因子(TNF) 大腸桿菌 細(xì)胞增殖 多克隆抗體　出處：《南京師范大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 人可溶性增殖誘導(dǎo)配體(hsAPRIL)是TNF家族的一個(gè)較新的成員，它與人B淋巴細(xì)胞刺激因子(hBAFF)同屬腫瘤壞死因子超家族的成員，且在該家族中兩者最為接近。本文從人外周血液總RNA中克隆出hsAPRIL cDNA，利用pET-30a這一原核表達(dá)載體成功構(gòu)建并表達(dá)了hsAPRIL蛋白，在對(duì)其進(jìn)行變復(fù)性后得到了其活性蛋白，并進(jìn)行細(xì)胞活性鑒定，證明所得到的確為其活性形式。另一方面以APRIL蛋白為抗原免疫實(shí)驗(yàn)兔，通過心臟取血從血清中得到其抗體，并通過飽和硫酸銨沉淀法以及Protein-A凝膠柱純化該抗體，利用western blotting鑒定抗體，后以ELISA來測(cè)定該多抗的效價(jià)。實(shí)驗(yàn)設(shè)計(jì)及結(jié)果如下： 1 hsAPRIL cDNA基因的釣取，，蛋白的表達(dá)、純化及活性鑒定 利用RT-PCR技術(shù)從健康人新鮮外周血中釣出增殖誘導(dǎo)配體基因，將其克隆于原核表達(dá)載體后，在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)目的蛋白，并通過Ni-IDA親和層析柱對(duì)該蛋白進(jìn)行純化，純化后的蛋白通過透析變復(fù)性后得到有活性蛋白，再利用其促進(jìn)腫瘤細(xì)胞增殖這一特性來鑒定其活性。 2 hsAPRIL多克隆抗體的制備及純化 將已制備好的抗原每隔10-15天免疫試驗(yàn)兔，在第三次免疫后從兔耳靜脈取血，測(cè)其效價(jià)。在確定多抗確有其特異性后從心臟取血得到抗血清。在利用飽和(NH4)SO4沉淀法將抗血清初步純化后再利用Ptotein-A親和層析柱對(duì)其進(jìn)一步純化。純化產(chǎn)物用western blotting以及ELISA檢測(cè)和測(cè)定其效價(jià)。
[Abstract]:Human soluble proliferation-inducing ligand hsAPRIL is a new member of the TNF family. It belongs to the tumor necrosis factor superfamily with human B lymphocyte stimulating factor hBAFF. HsAPRIL cDNA was cloned from the total RNA of human peripheral blood fluid. The prokaryotic expression vector pET-30a was used to construct and express the hsAPRIL protein successfully. After renaturation, the active protein was obtained and the cell activity was identified. On the other hand, the APRIL protein was used as antigen to immunize rabbits, and their antibodies were obtained from the serum by taking blood from the heart. The antibody was purified by saturated ammonium sulfate precipitation and Protein-A gel column, and the antibody was identified by western blotting. The titer of the polyclonal antibody was determined by ELISA. The experimental design and results are as follows: 1 hsAPRIL cDNA gene capture, protein expression, purification and activity identification The proliferation-inducing ligand gene was isolated from fresh peripheral blood of healthy people by RT-PCR technique and cloned into prokaryotic expression vector to induce the expression of target protein in E. coli BL21DE3. The protein was purified by Ni-IDA affinity chromatography. The purified protein was renatured by dialysis to obtain the active protein. The activity of the purified protein was identified by its ability to promote the proliferation of tumor cells. Preparation and purification of 2 hsAPRIL polyclonal antibody The rabbits were immunized with the prepared antigen every 10-15 days, and the blood was taken from the rabbit ear vein after the third immunization. Determination of the titer of polyantibodies. After determination of the specificity of polyantibodies, blood was taken from the heart to obtain antiserum. The antiserum was initially purified by SO4 precipitation and further purified by Ptotein-A affinity chromatography. The purified product was purified by western. Blotting and ELISA were used to detect and determine its titer.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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本文編號(hào)：1442311