【摘要】 目的 :构建鸟氨酸脱羧酶 (ODC)表达载体 ,表达ODC重组蛋白 ,制备其多克隆抗体并观察其在大肠癌诊断中的意义。方法 :从大肠癌组织中提取总RNA ,反转录PCR法扩增全长ODCcDNA ,将扩增的基因插入表达载体 pQE 30的BamHI和SalI酶切位点之间 ,DNA测序验证插入片段。将重组载体转化入宿主菌M 15中进行诱导表达 ,产生含 6 His tag的融合蛋白。WesternBlotting法检测表达蛋白。亲和层析法纯化融合蛋白 ,以该蛋白免疫小鼠制备多抗 ,免疫组化检测大肠癌组织中ODC的表达水平。结果 :酶切鉴定和DNA测序显示ODC重组表达载体中含有人ODCcDNA全长序列 ,与NIH基因库的人ODCcDNA比较有 99%的同源性。将该重组载体转化入大肠杆菌M 15中表达所得蛋白经WesternBlotting验证为目的蛋白。纯化融合蛋白 ,免疫小鼠获得多抗效价较高。免疫组化检测大肠癌组织中ODC的表达显示该抗体检测效果较好 ,大肠癌组织中ODC的表达水平明显高于正常组织 (P <0 .0 5 )。结论 :成功地构建了ODC表达载体、表达出ODC重组蛋白、制备出ODC多克隆抗体 ,为进一步研究结直肠癌的发病机理及其诊断和治疗方法打下了良好基础。更多还原

【Abstract】 Objective:To prepare a kind of human ODC polyclonal antibodies using an expressed ODC recombinant protein and explore its potential usage in early diagnosis of colorectal carcinoma.Methods:Total RNA was extracted from colon cancer tissues and amplified by reverse transcription PCR with two primers,which span the whole coding region of ODC.The synthesized ODC cDNA was cloned into vector pQE 30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30 ODC.The sequence of inserted fragment was confirmed by DNA sequencing,the fusion protein including 6His tag was purified by Ni NTA chro matographic column.Prepare polyclonal antibodies using the fusion protein as antigen and the expression of ODC in colorectal carcinoma tissues was assayed by the antibodies.Results:ODC expression vector was constructed and confirmed with restriction enzyme digestioin and subsequent DNA sequencing.The DNA sequence matching on NCBI Blast showed 99% homology.The vector was transformed into E.coli M15 and expressed.The expressed and purified ODC protein was verified with Western blotting.ELISA showed the antibodies have high affinity with ODC.Immunochemistry assay exhibit that Colorectal cancer tissues have higher concentration than the normal controls(P<0.05).Conclusion:The human ODC fusion protein is constructed expressed and purified in vitro.Use it as antigen to prepare polyclonal antibodies.The later was used in immunochemistry which showed ODC concentration is very high in colorectal carcinoma.更多还原