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
冯雳 1卢静 2魏思雯 2胡长峰 1弋美霞 2罗仕超2
作者信息
- 1. 江汉大学医学部,武汉 430056;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
- 2. 江汉大学生命科学学院,武汉 430056
- 折叠
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∶512 000,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.
Abstract
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∶512 000 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Key words
Chlamydomonas reinhardtii/leucine-rich repeat/innate immunity/defense response/polyclonal antibody引用本文复制引用
出版年
2024
NETL
NSTL
万方数据