莱茵衣藻LRR-1蛋白多克隆抗体的制备与鉴定  

Preparation and identification of polyclonal antibody against LRR-1 protein of Chlamydomonas reinhardtii

作　　者：冯雳 卢静 魏思雯 胡长峰[1,2] 弋美霞 罗仕超 FENG Li;LU Jing;WEI Siwen;HU Zhangfeng;YI Meixia;LUO Shichao(School of Medicine,Jianghan University,Wuhan 430056,China;Hubei Key Laboratory of Environmental and Health Effects of Persistent Toxic Substances,Jianghan University,Wuhan 430056,China;College of Life Sciences,Jianghan University,Wuhan 430056,China)

机构地区：[1]江汉大学医学部,武汉430056 [2]江汉大学持久性有毒污染物环境与健康危害湖北省重点实验室,武汉430056 [3]江汉大学生命科学学院,武汉430056

出　　处：《华中农业大学学报》2024年第5期297-302,共6页Journal of Huazhong Agricultural University

基　　金：国家重点研发计划项目(2020YFA0907400);国家自然科学基金项目(32170702)。

摘　　要：为深入研究富含亮氨酸重复序列(leucine-rich repeat,LRR)在机体先天免疫过程中的作用机制,以莱茵衣藻(Chlamydomonas reinhardtii)为模式生物,通过生物信息学预测分析CrLRR-1蛋白的二级结构、亲水性及免疫原性等理化性质,人工设计并合成特异性肽段,与钥孔血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联后免疫新西兰白兔。结果显示:采用间接ELISA法测定抗血清效价达到1∶512000,将抗血清依次经Protein A和抗原亲和纯化后得到CrLRR-1蛋白多克隆抗体;利用免疫印迹与免疫荧光检测制备的抗体,该抗体可特异性识别莱茵衣藻内源LRR-1蛋白,且CrLRR-1蛋白主要定位于细胞膜。结果表明,成功制备CrLRR-1蛋白多克隆抗体。Chlamydomonas reinhardtii was used as a model organism to deeply study the function mechanism of leucine rich repeat(LRR)in the process of innate immunity in the organism.The physico⁃chemical properties of CrLRR-1 protein including secondary structure,hydrophilicity,and immunogenicity were predicted and analyzed with bioinformatics.The CrLRR-1 specific peptide was artificially designed and synthesized,then coupled with keyhole limpet hemocyanin(KLH)to immunize New Zealand white rab⁃bits.The results showed that the antiserum titer was determined to be 1∶512000 by indirect ELISA.The CrLRR-1 protein polyclonal antibodies were obtained after sequentially purifying the antiserum with Protein A and antigen affinity.The antibodies prepared with immunoblotting and immunofluorescence detection spe⁃cifically recognized the endogenous LRR-1 protein in Chlamydomonas reinhardtii,and the CrLRR-1 pro⁃tein was mainly localized on the cell membrane.It is indicated that the polyclonal antibody against CrLRR-1 protein were successfully prepared and identified.

关 键 词：莱茵衣藻 富亮氨酸重复序列 先天免疫 防御反应 多克隆抗体 

分 类 号：Q786[生物学—分子生物学]