抗人CLC-7多克隆抗体的制备  

The preparation of anti-CLC-7 polyclonal antibody

作　　者：徐勇[1] 王瑶[1] 袁园 赵朔暄 赵文然[1] Xu Yong;Wang Yao;Yuan Yuan;Zhao Shuoxuan;Zhao Wenran(Department of Cell biology, Harbin Medical University, Harbin 150081, China)

机构地区：[1]哈尔滨医科大学细胞生物学教研室,150081

出　　处：《国际免疫学杂志》2019年第2期127-130,共4页International Journal of Immunology

基　　金：黑龙江省卫生计生委科研课题(2007-455).

摘　　要：目的制备抗氯离子通道蛋白(chloride channel 7,CLC-7)多克隆抗体.方法将表达载体pEGFP-CLC-7导入感受态大肠杆菌DH-5α,利用异丙基β-D-硫代半乳糖苷(isopropyl-β-d-thiogalacto-side,IPTG)诱导细菌表达CLC-7蛋白;经SDS-PAGE电泳后切胶获得纯化的CLC-7蛋白,与免疫佐剂乳化后免疫新西兰大白兔,免疫5次后,经心脏采血获取抗血清,用Western blot检测抗血清特异性及效价.结果与市售的CLC-7抗体比较,所获抗CLC-7抗血清可识别细菌及HeLa细胞表达的CLC-7蛋白,表明抗血清有较好的特异性;1:4000倍稀释抗血清时,Western blot实验仍可获得清晰的反应带,表明所制备的抗CLC-7多克隆抗体有较高的效价.结论利用原核表达的CLC-7蛋白作为抗原免疫动物,获得了高效价且特异性好的抗人CLC-7蛋白多克隆抗体,该抗体可用于后续研究.Objective To prepare the polyclonal antibody against human chloride channel 7(CLC-7). Methods EGFP-CLC-7 fusion protein was expressed in E. coli by transforming the bacteria DH-5α with the construct pEGFP-CLC-7. The expression was induced by IPTG for 24 h. Bacterial proteins were extracted and subjected to SDS-PAGE electrophoresis. The protein of interest was identified on the gel of SDS-PAGE according to the molecular weight. The gel containing the separated protein of the interest was collected,minced,and mixed with immune adjuvant BCG. New Zealand white rabbits were immunized with 1g protein extract mixed with BCG for five times in 6 weeks. Sera of the immunized rabbits were collected. The specificity and the titer of the anti-body against CLC-7 were determined by Western blot. Results The prepared antibody against human CLC-7 showed the same pattern in the reaction with CLC-7 expressed in both E. coli and HeLa cells as the commercial-ly available CLC-7 polyclonal antibody(obtained from Abcam). The titer of the prepared polyclonal antibody a-gainst CLC-7 reached to 1: 4000. Conclusion Polyclonal antibody against human CLC-7 was obtained by im-munizing rabbit with CLC-7 expressed in E. coli. This antibody,with titration up to 1: 4000,showed high speci-ficity compared to the commercially available CLC-7 antibody.

关 键 词：氯离子通道蛋白7 抗体 

分 类 号：R392[医药卫生—免疫学]