尿激酶受体在大肠杆菌中的表达及其多克隆抗体的制备  

EXPRESSION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN E.COLI. AND PREPARATION OF uPAR POLYANTIBODY

作　　者：唐辉滨[1,2,3] 于伟英[1,2,3] 何诚[1,2,3] 廖劲辉 田昱 郑起[1,2,3] 朱运松 

机构地区：[1]上海医科大学基础医学院分子遗传研究室 [2]中山医院肝癌研究所 [3]中国科学院上海生命科学中心

出　　处：《上海医科大学学报》1998年第3期185-188,共4页Journal of Fudan University(Medical Science)

基　　金：国家自然科学基金

摘　　要：目的构建尿激酶受体（ｕｒｏｋｉｎａｓｅ－ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｒｅｃｅｐｔｏｒ，ｕＰＡＲ）表达质粒并在大肠杆菌中表达，用纯化的表达产物ｕＰＡＲ蛋白制备多克隆抗体并初步应用于肿瘤标本的检测。方法用基因工程的方法构建ｕＰＡＲｃＤＮＡ表达质粒ｐＬＹ４－ｕＰＡＲ，转化重组大肠杆菌在４２℃条件下诱导表达；利用ＳＤＳ－ＰＡＧＥ进行纯化并用纯化后的抗原免疫新西兰兔制备多克隆抗体，应用制备的抗体对乳腺癌和肝癌中ｕＰＡＲ的分布进行检测。结果ｕＰＡＲ表达质粒在大肠杆菌中高效表达，表观Ｍｒ为３０×１０３左右。表达的ｕＰＡＲ占全菌蛋白的２０％左右；获得了效价为１∶３２的ｕＰＡＲ多克隆抗体；发现ｕＰＡＲ主要分布在癌细胞的表面及胞质内，而在癌旁组织中则较弱。结论ｕＰＡＲ在大肠杆菌获得高效表达，并制备得到多克隆抗体。ｕＰＡＲ在癌组织（乳腺癌及肝癌）与癌旁组织间的表达差异提示可将其应用到临床肿瘤的诊断与肿瘤转移的实验性研究中。URPOSE To Clone and express urokinasetype plasminogen activator receptor(uPAR) cDNA in E. coli. and prepare the uPAR antiserum for further study.METHODS uPAR expression plasmid pLY4-uPAR was constructed by means of recombinant DNA technics and transformed into E.coli. JF1125. uPAR protein was synthesized at 42℃ and identified by Wes￣tern blot. After purified by SDS-PAGE, it was used to immunize Newzealand rabbits to acquire uPAR antiserum. With immunohistochemistry, the antiserum was also used to detect the distribution of uPAR in breast cancer and liver cancer.RESULTS The uPAR protein was expressed by the host cell, existing in the form of inclusion body with molecular weight around 30×103. The amount of the recombinant protein was estimated as 20% of the whole bacterial protein; the titre of the antiserum was up to 1∶32. In addition, immunostaining showed that uPAR was mainly on the cell surface and in the cytoplasm in breast cancer cells and liver cancer cells. However, the staining was weak in the tissues near the mammary tumor nest and the liver hemangioma.CONCLUSIONS High level of expression of uPAR was obtained in E.coli. The purified uPAR protein from recombinant bacteria can be applied as antigen to immunize rabbits to get high titre uPAR antiserum.

关 键 词：尿激酶受体 多克隆抗体 大肠杆菌 表达 肿瘤血管 

分 类 号：R730.59[医药卫生—肿瘤]