贵州医科大学学报

2015, v.40;No.180(09) 954-958

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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

张丽娟;高晓明;
ZHANG Lijuan;GAO Xiaoming;Institute of Biology and Medicine Sciences,Soochow University;

摘要(Abstract):

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 immunobiological 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(KeyWords): 免疫学;钙网蛋白;蛋白片段;生物学活性
immunology;calreticulin;protein fragment;biological activity

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作者(Author): 张丽娟;高晓明;
ZHANG Lijuan;GAO Xiaoming;Institute of Biology and Medicine Sciences,Soochow University;

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DOI: 10.19367/j.cnki.1000-2707.2015.09.016

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