牛脑钙调素的分离纯化、鉴定及其免疫原制备

[目的]从牛脑中分离纯化钙调素(CaM),并进行生化鉴定,同时用碳二亚胺法制备CaM偶联免疫原.[方法]以phenyl-Sepharose层析法从牛脑中分离纯化CaM,用PDE法检测其活性,以SDS-PAGE、PAGE检测其分子量并观察CaM有无Ca~(2+)结合时的电泳行为,检测其紫外扫描光谱,同时用交联剂碳二亚胺制备CaM偶联抗原进行动物免疫,并用间接Elisa法检测动物血清免疫效价.[结果]纯化后的CaM对PDE有明显的激活作用,电泳结果显示为均一条带,分子量约为17.3 kDa,在SDS-PAGE中,CaM结合Ca~(2+)时比未结合Ca2+时迁移率快;而在PAGE中,迁移率在结合Ca~(2+)比未结合Ca~(2+)时慢;紫外光谱扫描显示约在251、260、267、272 和283 nm有5个吸收峰,动物血清免疫效价为1∶800.[结论]用phenyl-Sepharose层析法纯化CaM和用碳二亚胺法制备CaM偶联抗原是成功的.

Isolation,Purification and Identification of Bovine Brain Calmodulin and Preparation of Antigen

[Objective]Calmodulin(CaM) was isolated and purified from bovine brain and identified with biochemistry, while CaM-couple antigen was prepared with EDC method.[Method]Calmodulin was isolated and purified from bovine brain by phenyl-Sepharose hydrophobic chromatography column.Its activity was detected by PDE method, the molecular weight was tested by SDS-PAGE, PAGE and electrophoresis behavior was observed, at the same time, its ultraviolet scanning spectrum were detected.Then CaM-couple antigens, prepared with cross linking agent-EDC, were used for animal immunization, and finally evaluated animal serum immunopotency by indirect Elisa.[Result]Cyclic nucleotide phosphodiesterase (PDE) can be activated by CaM, the results of electrophoresis showed to be homogeneous, and molecular weight was 17.3 KDa. The transport ratio of CaM-Ca~(2+) was faster than CaM-EGTA in SDS-PAGE, while the result was opposite in PAGE. The results of ultraviolet absorption-spectra showed there were 5 absorption peak at about 251 nm,260 nm,267 nm,272 nm and 283 nm. Then, animal serum can be purified by phenyl-Sepharose hydrophobic chromatography column and CaM-couple antigen is prepared by EDC method.