基因工程链激酶(r-SK)纯化及其单克隆抗体制备和鉴定  

Purification of Recombinant Streptokinase (r-SK) and Preparation and Characterization of Anti-r-SK Monoclonal Antibody

作　　者：丁皓[1] 朱运松[1] 吴晓俐[1] 周兴华 宋后燕[1] 

机构地区：[1]上海医科大学分子遗传学研究室,上海200032

出　　处：《生物工程学报》1994年第1期56-60,共5页Chinese Journal of Biotechnology

基　　金：国家"85"攻关课题(85-722-01-06)一部分

摘　　要：采用Ｒｏｔｏｆｏｒ等电聚聚焦和分子筛技术纯化大肠杆菌表达的重组链激酶（ｒ－ＳＫ），其纯度为９７％，比活性１×１０＾６ＩＵ／ｍｇ，回收率４１％，分析所纯化的ｒ－ＳＫ Ｎ端氨基酸顺序证实其１－１５个氨基酸顺序与ＳＫ基因的核苷酸顺充所示３联密码子一致。以纯化ｒ－ＳＫ为抗原，通过杂交瘤技术构建了分泌抗ｒ－ＳＫ单克隆抗体杂交瘤细胞。该ＭｃＡｂ属ＩｇＧ１亚型。Isoelectric focusing in the Rotofor cell combined with molecular sieve chro-matography was used for the purification of the recombinant streptokinase (r-SK) expression in E. colt. The purity and special activity of r-SK were 97%, 1×105IU/mg, respectively. The recovery of this method was 41%. Analyses of N-terminal amino acid of purified r-SK comfirmed that the residue 1 to residue 15 was identical to the codens showed by the nucleotide of SK gene. A clone (4D11) producing monoclonal antibody (McAb) against SK was abtained by fusion of mouse myeloma cells (SP2/0) and spleen cells previously immunised with purified-r-SK. The McAb was IgG1 subtype and specially reacted with r-SK and SK from group C of Streptococcus B-hemolyticus. The McAb also inhibited the function of SK as a plasminogen activator determined by chromogenic assay. Western blot comfirmed that the McAb could recognized MWtl6 250 Da fragment of SK clevaged with CNBr. It was proposed that residue 71 to 237 of SK may participate in binding of SK and plasminogen.

关 键 词：重组 链激酶 等电聚焦 单克隆抗体 

分 类 号：R394.8[医药卫生—医学遗传学]