BAFF受体TACI融合蛋白的表达纯化与功能研究
发布时间：2018-07-13 19:16
【摘要】：B细胞活化因子(B cell activating factor,BAFF)属于肿瘤坏死因子超家族的一员。它是外周B细胞存活的重要因子。在动物模型中,BAFF的过表达会导致自身免疫病样症状的发生。研究发现,在不同类型的自身免疫性疾病患者的血清中,BAFF的含量是明显升高的。近期研究还发现BAFF还与部分肿瘤疾病的发生有关。因此,BAFF的拮抗剂可能成为治疗自身免疫疾病和某些肿瘤的新型药物。 由于BAFF和受体TACI具有高亲和力,可特异性识别BAFF并与之结合,因此体外构建表达的TACI可中和体内过表达的BAFF,抑制B细胞的增殖和活化,缓解和治疗与BAFF相关的自身免疫病和B细胞肿瘤。本课题的研究为开发BAFF拮抗剂治疗BAFF相关疾病提供了实验基础。 本课题研究方法:对BAFF受体TACI融合蛋白原核表达与纯化,在体外研究TACI对B系淋巴瘤细胞增殖的影响,并初步分析了TACI抑制肿瘤细胞系增殖的机理。 主要研究结果: (一)TACI融合蛋白原核表达纯化:本实验室已将钓取的TACI基因片段连接到原核表达载体pET-32a上。将重组正确的质粒转化大肠杆菌Rosetta,获得TACI表达工程菌。经IPTG诱导,融合蛋白利用镍金属螯合琼脂糖凝胶亲和层析柱纯化。 (二)TACI抑制B系淋巴瘤细胞系细胞的增殖:某些B系淋巴瘤细胞系表达BAFF及其受体,并且通过自分泌BAFF来促进肿瘤细胞的增殖。可溶性TACI蛋白能够通过特异性阻断BAFF抑制B系淋巴瘤细胞系的增殖,并且具有剂量依赖性。 (三)TACI抑增殖的机理:TACI能够将Daudi细胞阻滞在DNA合成期(即S期) ,而且具有一定的剂量依赖性。TACI对Daudi细胞增殖抑制作用,主要是通过调控细胞周期,而非凋亡达到抑增殖效果。
[Abstract]:B cell activating factor (B cell activating factor) belongs to the tumor necrosis factor superfamily. It is an important factor in the survival of peripheral B cells. Overexpression of BAFF may lead to autoimmune disease-like symptoms in animal models. Serum BAFF levels were significantly increased in patients with different types of autoimmune diseases. Recent studies have also found that BAFF is also associated with some tumor diseases. Therefore, BAFF antagonist may be a new drug for autoimmune diseases and some tumors. Because BAFF and TACI have high affinity, BAFF can be specifically recognized and combined with BAFF, so TACI can neutralize the overexpression of BAFF in vivo and inhibit the proliferation and activation of B cells. To relieve and treat BAFF associated autoimmune diseases and B cell tumors. This study provides experimental basis for the development of BAFF antagonist in the treatment of BAFF related diseases. Methods: the prokaryotic expression and purification of BAFF receptor TACI fusion protein were studied in vitro to study the effect of TACI on the proliferation of B-lineage lymphoma cells and the mechanism of TACI inhibiting the proliferation of tumor cell lines was preliminarily analyzed. The main results were as follows: (1) expression and purification of TACI fusion protein: the TACI gene fragment was ligated to the prokaryotic expression vector pET-32a in our laboratory. The recombinant plasmid was transformed into E. coli Rosetta to obtain TACI expression engineering bacteria. After induced by IPTG, the fusion protein was purified by nickel metal chelate agarose gel affinity chromatography. (2) TACI inhibits the proliferation of B-lineage lymphoma cell lines: some B-lineage lymphoma cell lines express BAFF and its receptors, and promote the proliferation of tumor cells by autocrine BAFF. Soluble TACI protein can inhibit the proliferation of B line lymphoma cell line by blocking BAFF in a dose-dependent manner. (3) Taci inhibited the proliferation of Daudi cells by blocking Daudi cells in DNA synthesis phase (S phase) in a dose-dependent manner. The inhibitory effect of TACI on the proliferation of Daudi cells was mainly achieved by regulating cell cycle, but not by apoptosis.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：2120472
[Abstract]:B cell activating factor (B cell activating factor) belongs to the tumor necrosis factor superfamily. It is an important factor in the survival of peripheral B cells. Overexpression of BAFF may lead to autoimmune disease-like symptoms in animal models. Serum BAFF levels were significantly increased in patients with different types of autoimmune diseases. Recent studies have also found that BAFF is also associated with some tumor diseases. Therefore, BAFF antagonist may be a new drug for autoimmune diseases and some tumors. Because BAFF and TACI have high affinity, BAFF can be specifically recognized and combined with BAFF, so TACI can neutralize the overexpression of BAFF in vivo and inhibit the proliferation and activation of B cells. To relieve and treat BAFF associated autoimmune diseases and B cell tumors. This study provides experimental basis for the development of BAFF antagonist in the treatment of BAFF related diseases. Methods: the prokaryotic expression and purification of BAFF receptor TACI fusion protein were studied in vitro to study the effect of TACI on the proliferation of B-lineage lymphoma cells and the mechanism of TACI inhibiting the proliferation of tumor cell lines was preliminarily analyzed. The main results were as follows: (1) expression and purification of TACI fusion protein: the TACI gene fragment was ligated to the prokaryotic expression vector pET-32a in our laboratory. The recombinant plasmid was transformed into E. coli Rosetta to obtain TACI expression engineering bacteria. After induced by IPTG, the fusion protein was purified by nickel metal chelate agarose gel affinity chromatography. (2) TACI inhibits the proliferation of B-lineage lymphoma cell lines: some B-lineage lymphoma cell lines express BAFF and its receptors, and promote the proliferation of tumor cells by autocrine BAFF. Soluble TACI protein can inhibit the proliferation of B line lymphoma cell line by blocking BAFF in a dose-dependent manner. (3) Taci inhibited the proliferation of Daudi cells by blocking Daudi cells in DNA synthesis phase (S phase) in a dose-dependent manner. The inhibitory effect of TACI on the proliferation of Daudi cells was mainly achieved by regulating cell cycle, but not by apoptosis.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
【共引文献】
相关期刊论文 前2条
1 于孟学;曹金;李薇;;生物制剂治疗系统性红斑狼疮的新进展[J];北京医学;2008年01期
2 颜克香;项蕾红;郑志忠;Stuart Mardim;;2010年后皮肤科的主要治疗进展[J];中国中西医结合皮肤性病学杂志;2011年01期
相关硕士学位论文 前1条
1 张小丹;TACI-Ig对ConA活化淋巴细胞的染色质诱导SLE样小鼠模型的治疗作用[D];安徽医科大学;2011年
，本文编号：2120472
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2120472.html
最近更新
教材专著
