人可溶性增殖诱导配体的表达、纯化、活性鉴定以及抗体的制备

发布时间：2018-01-19 03:00

本文关键词： 人增殖诱导配体(APRIL) 肿瘤坏死因子(TNF) 大肠杆菌细胞增殖多克隆抗体　出处：《南京师范大学》2007年硕士论文　论文类型：学位论文

【摘要】： 人可溶性增殖诱导配体(hsAPRIL)是TNF家族的一个较新的成员，它与人B淋巴细胞刺激因子(hBAFF)同属肿瘤坏死因子超家族的成员，且在该家族中两者最为接近。本文从人外周血液总RNA中克隆出hsAPRIL cDNA，利用pET-30a这一原核表达载体成功构建并表达了hsAPRIL蛋白，在对其进行变复性后得到了其活性蛋白，并进行细胞活性鉴定，证明所得到的确为其活性形式。另一方面以APRIL蛋白为抗原免疫实验兔，通过心脏取血从血清中得到其抗体，并通过饱和硫酸铵沉淀法以及Protein-A凝胶柱纯化该抗体，利用western blotting鉴定抗体，后以ELISA来测定该多抗的效价。实验设计及结果如下： 1 hsAPRIL cDNA基因的钓取，，蛋白的表达、纯化及活性鉴定利用RT-PCR技术从健康人新鲜外周血中钓出增殖诱导配体基因，将其克隆于原核表达载体后，在大肠杆菌BL21(DE3)中诱导表达目的蛋白，并通过Ni-IDA亲和层析柱对该蛋白进行纯化，纯化后的蛋白通过透析变复性后得到有活性蛋白，再利用其促进肿瘤细胞增殖这一特性来鉴定其活性。 2 hsAPRIL多克隆抗体的制备及纯化将已制备好的抗原每隔10-15天免疫试验兔，在第三次免疫后从兔耳静脉取血，测其效价。在确定多抗确有其特异性后从心脏取血得到抗血清。在利用饱和(NH4)SO4沉淀法将抗血清初步纯化后再利用Ptotein-A亲和层析柱对其进一步纯化。纯化产物用western blotting以及ELISA检测和测定其效价。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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