期刊文献+

环加氧酶2基因的原核表达、抗体制备及其在肝癌中的表达 被引量:1

Polyantibady Preparation of Cyclooxygenase-2 Protein and Its Expression Analysis in Hepatocellular Carcinoma
下载PDF
导出
摘要 用PCR技术扩增环加氧酶2(COX-2)基因的C末端777 bp的片段,插入到pET-28b(+)中,构建原核表达载体.转入大肠杆菌BL21(DE3),经IPTG诱导产生包涵体形式his-COX-2融合蛋白.用镍离子螯合柱(Ni-NTA)纯化融合蛋白,获得纯度较高的原核表达蛋白.纯化后的蛋白免疫家兔制备多克隆抗体,用ELISA及West-ern blotting分别检测抗体.用此抗体Western blotting检测肝癌组织中的环加氧酶2的表达.ELISA及Westernblotting结果显示免疫家兔所得的环加氧酶2抗体具有很高的效价,并能够特异性的识别真核细胞中的环加氧酶2.用此抗体检测肝癌组织中的环加氧酶2的表达,证实肝癌组织中伴随有环加氧酶2的大量表达.同时所得的环加氧酶2抗体具有很高的效价和特异性,为进一步研究环加氧酶2的功能提供了条件. Expression vector carrying cyclooxygenase 2 (COX-2) gene was constructed by inserting the C terminal 777 bp fraction amplified by PCR into the vector pET 28b(+). The fusion protein was expressed in the form of inclusion body after induction by IPTG in BL21 (DE3). Using Ni NTA chelating column, the fusion protein was highly purified. The polyantibody was prepared by immunizing the rabbits with five injections of the purified protein. ELISA and Western Blotting were performed to test its specificity and sensitivity. Meanwhile, cyclooxygeanse-2 expression was detected in hepatocellular carcinoma and surrounding noncancerous liver tissues by Western Blotting using the antiserum. The polyantibody can recognize cyclooxygease-2 with high specificity and sensitivity and cyclooxygenase 2 was expressed at higher level in hepatocellular carcinoma compared to that in surrounding noncancerous liver tissues. Preparation of cyclooxygeanse-2 polyantlbody make it possible to further study the biological functions.
出处 《武汉大学学报(理学版)》 CAS CSCD 北大核心 2006年第2期197-201,共5页 Journal of Wuhan University:Natural Science Edition
基金 国家自然科学基金资助项目(30570066)
关键词 环加氧酶2(COX-2) 原核表达 抗体制备 肝癌 cyclooxygenase-2 gene prokaryotic expresslon antiserum preparation hepatocellular carcinoma
  • 相关文献

参考文献6

二级参考文献99

  • 1潘赛英,胡跃,张苏展,陈丽荣,王海军.环氧化酶-2表达与大肠癌TNM分期、分化及转移之间的关系[J].中华肿瘤杂志,2004,26(5):300-300. 被引量:5
  • 2詹俊,刘建平,朱兆华,姚和瑞,陈春燕.Relationship between COX-2 expression and clinicopathological features of colorectal cancers[J].Chinese Medical Journal,2004(8):1151-1154. 被引量:24
  • 3CLARK E D B.Protein refolding for industrial processes[J]. Current Opinion in Biotechnology,2001,12:202-207. 被引量:1
  • 4KOHNO T,CARMICHAEL D F,SOMMER A,et al.Refolding of recombinant proteins[J]. Methods Enzymol,1990,185:187-195. 被引量:1
  • 5VAN KIMMENADE A,BOND M W,SCHUMACHER J H,et al.Expression,renaturation and purification of recombinant human Interleukin 4 from Escherichia coli[J].Eur J Biochem,1988,173:109-114. 被引量:1
  • 6CLARK E D B.Refolding of recombinant proteins[J]. Curr Opin Biotechnol,1998,9(2):157-163. 被引量:1
  • 7MAEDA Y,UEDA T,IMOTO T.Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone[J].Protein Eng,1996,9(1):95-100. 被引量:1
  • 8HWANG H S,CHUNG H S.Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body[J]. Protein Expr Purif,2002,25(3):541-546. 被引量:1
  • 9WEST S M,CHAUDHUI J B,HOWELL J A.Improved protein refolding using hollow-fibre membrane dialysis[J]. Biotechnol Bioeng,1998,57(5):590-599. 被引量:1
  • 10FAHEY E M,CHAUDHURI J B,BINDING P.Refolding and purification of urokinase plaminogen activator fragment by chromatography[J]. J Chromatogr B Biomed Sci Appl,2000,737(1-2):225-235. 被引量:1

共引文献106

同被引文献13

  • 1车婧,韩金祥,王世立.促进包涵体蛋白复性的几种有效添加剂[J].医学分子生物学杂志,2004,1(2):122-125. 被引量:14
  • 2邢丽华,张珍祥,徐永健,张惠兰,刘剑波.环氧化酶-2在肺癌血管生成中的作用及机制[J].华中科技大学学报(医学版),2005,34(2):189-191. 被引量:3
  • 3杨华强,张荣环,章正华,万楚成,夏云金.COX-2和Survivin在B细胞非霍奇金淋巴瘤中的表达及意义[J].四川大学学报(医学版),2007,38(4):731-733. 被引量:2
  • 4[2]Mark A H.Cyclooxygenase-2:How Good is it as a Target for Cancer Chemoprevention?[j].European Journal of Cancer,2005,41:1854-1863. 被引量:1
  • 5[3]Appleby S B,Ristimaki A,Neilson K,et al.Structure of the Human cyclooxygenase-2 Gene[J].Journal of Biochemistry,1994,302(3):723-727. 被引量:1
  • 6[7]Thun M J,Namboodiri M M,Heath C W Jr.Aspirin Use and Reduced Risk of Fatal Colon Cancer[J].New England Journal of Medcine,1991,325(23):1593-1596. 被引量:1
  • 7[8]Oshima M,Dinchuk J E,Kargman S L,et al.Suppression of Intestinal Polyposis in APCA7 16 Knockout Mice by Inhibition of Prostaglandin Endoperoxide synlhase-2(COX-2)[J].Cell,1996,87(5):803-809. 被引量:1
  • 8[9]Dalwadi H,Sharma S,Pold M,et al.Cyclooxygenase 2-Dependent Expression of Survivin is Critical for Apoptosis Resistance in Non-Small Cell lung Cancer[J].Cancer Research,2004,64(18):6359-6362. 被引量:1
  • 9[10]Costa C,Soares R,Reis-Filho J S,et al.Cyclo-Oxygenase 2 Expression is Associated with Angiogenesis and Lymph Node metastasis in Human Breast Cancer[J].Journal of Clinical Pathology,2002,55(6):429-34. 被引量:1
  • 10[13]Ferrari V,Valcamonico F,Amoroso V,et al.Gemcitabine Plus Celecoxib(GECO)in Advanced Pancreatic Cancer:a Phase H Trial[J].Cancer Chemotherapy and Pharmacology,2005,2(57):185-190. 被引量:1

引证文献1

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部