钙网蛋白片段cDNA克隆、蛋白表达及免疫学活性研究  

A Study of cDNA Cloning,Protein Expression and Immunological Activity of Calreticulin Fragment

作　　者：张丽娟[1] 高晓明[1] 

机构地区：[1]苏州大学生物医学研究院,江苏苏州215123

出　　处：《贵阳医学院学报》2015年第9期954-958,共5页Journal of Guiyang Medical College

摘　　要：目的:研究钙网蛋白(CRT)发挥免疫生物学活性的主要功能区域。方法:采用PCR法从CRT基因cDNA全长中扩增第150-230位编码氨基酸所对应的核苷酸序列片段,利用分子生物学方法构建原核重组表达质粒,采用大肠杆菌表达系统进行原核表达;通过亲和纯化得到目的蛋白,分析目的蛋白特性;并通过体内、体外实验对蛋白的免疫学活性进行分析。结果:成功构建了表达CRT150-230片段(rCRT/150-230)的原核表达质粒,并从细菌裂解液上清中纯化得到目的蛋白,蛋白单体通过分子间二硫键形成二聚体,天然状态下形成多聚体;体外研究表明rCRT/150-230促进小鼠脾细胞的分裂,活化巨噬细胞产生一氧化氮(NO),诱导人外周血单个核细胞产生TNF-α;体内研究表明,rCRT/150-230刺激小鼠产生高滴度抗体,该特异性抗体能识别rCRT/150-230与rCRT。结论:rCRT/150-230蛋白具有与rCRT相似的免疫活性,因此推测该蛋白片段是全长CRT发挥免疫学活性的区域。Objective： To investigate the major functional areas of calreticulin （CRT） that exerts im- munobiological activity. Methods： Amplified a 150 -230 fragment of rCRT and constructed a prokaryotic expression vector by molecular biology method. Then adopted E. Coli to express the protein which was purified by affinity purification. The biochemical property of rCRT/150 -230 was tested. Analyzing the immunobiological activity of this protein in vitro and in vivo. Results： rCRT/150 - 230 protein was purified from the bacterial lysate supernatant, and it had good solubility and existed as polymer in nature, rCRT/150-230 could effectively stimulate the proliferation of mice spleen cells, and activate the secretion of NO of mice macrophage, and stimulate human peripheral blood mononuclear cells to secrete TNF -α, and has strong immunogenicity that induces the process of humoral immune response in mice. Conclusion： rCRT/150 -230 appears similarity immunobiologic functions with rCRT, it could be inferred that it might be the major functional area of rCRT.

关 键 词：免疫学 钙网蛋白 蛋白片段 生物学活性 

分 类 号：R34-33[医药卫生—基础医学]