survivin蛋白的原核高效、可溶性表达及其抗体在恶性肿瘤早期诊断中的应用  

Value of anti-survivin in early diagnosis of malignant tumors

作　　者：吴素香[1] 马骢[2] 郭建巍[2] 黎卫平[1] 孔路科[1] 魏杰[1] 

机构地区：[1]兰州大学医学院病理学教研室,兰州730000 [2]海军总医院检验科,北京100037

出　　处：《第三军医大学学报》2008年第17期1641-1643,共3页Journal of Third Military Medical University

摘　　要：目的实现重组survivin在大肠杆菌中的高效、可溶性表达,检测健康人和肿瘤患者血清中anti-survivin并探讨其在肿瘤早期诊断中的应用价值。方法重组PET32a-survivin质粒转化BL21感受态细胞,IPTG诱导表达8h后对表达产物行溶菌酶处理、冻融及超声处理,SDS-PAGE及Westernblot鉴定后用镍离子金属螯合层析柱纯化。建立基于重组融合蛋白的survivin特异性抗体的检测方法,对300份健康血清、144份肿瘤血清anti-survivin进行了检测,并将anti-sur-vivin与肝癌、肠癌、胰腺癌、卵巢癌患者中AFP、CEA、CA199、CA125等肿瘤标志物进行了联合分析。结果重组survivin融合蛋白在BL21中获得了高效、可溶性表达,表达量约占菌体总蛋白的30%。建立了检测survivin抗体的间接ELISA方法,anti-survivin在不同的肿瘤血清中均有表达,阳性率各不相同。结论成功的实现了survivin蛋白在大肠杆菌中的高效、可溶性表达;结果提示在肿瘤的辅助诊断中,anti-survivin与现有的肿瘤标志物CA199、CEA、CA-199和CA-125联合检测,可以互相补充其不足,提高肿瘤的诊断率。Objective To establish a soluble expression of recombinant survivin in E. coli, and evaluate the role of anti-survivin in tumor diagnosis. Methods The recombinant survivin was screened by ampicillin resistance, identified by PCR and double digestion of endonucleases. The sequenced DNA of survivin was analyzed by BLAST. The survivin/Trx fusion protein expressed in E. coli BL21 （ DE3 ） was induced with IPTG, identified by Western blot and purified with Ni-NTA agarose respectively. The anti-survivin antibodies in serum were detected with an indirect ELISA. By the methods above, 144 serum samples from cancer patients and 300 serum samples from normal subjects were analyzed. Meanwhile, the joint detection of anti-survivin, AFP, CEA, CA199, CA125 in blood sera from cancer patients was detected. Results After PCR and double digestion with endonucleases, the survivin gene was inserted into the prokaryotic expression vector PET 32a （ ＋ ）. After DNA sequencing, the constructed expression plasmid was transformed into competent cells E. coli BL21 （DE3）. Western blot identified that the recombinant survivin/Trx fusion protein was specific against survivin antibody. The purity of the protein was over 96% after purified by Ni-NTA agarose. Anti-survivin was expressed in the sera of different cancer patients, but the positive rate varied. Conclusion Prokaryotic expression plasmid PET 32a（ ＋ ）/survivin was successfully constructed and highly purified survivin/Trx fusion protein was obtained. The detection results of anti-survivin, AFP, CEA, CA199, CA125 in blood serum show in the diagnosis of tumors, the joint detection can overcome the shortfall of each gene and enhance the diagnostic rate.

关 键 词：生存素 生存素抗体 酶联免疫吸附实验 重组融合蛋白 

分 类 号：R341[医药卫生—基础医学] R446