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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。
OBJECTIVE: To construct ornithine decarboxylase (ODC) expression vector and express ODC recombinant protein to prepare its polyclonal antibody and observe its significance in the diagnosis of colorectal cancer. METHODS: Total RNA was extracted from colorectal cancer tissues. The full length ODC cDNA was amplified by reverse transcription polymerase chain reaction (RT - PCR). The amplified gene was inserted into the BamHI and SalI restriction sites of the expression vector pQE30. DNA sequencing confirmed the inserted fragment. The recombinant vector was transformed into the host strain M15 for induction of expression, resulting in a 6 His tag-containing fusion protein. Western Blotting method to detect the expression of protein. The fusion protein was purified by affinity chromatography, and the polyclonal antibody was prepared by immunizing mice with this protein. The expression of ODC in colorectal cancer tissues was detected by immunohistochemistry. Results: The restriction endonuclease digestion and DNA sequencing showed that the ODC recombinant expression vector contained the full-length cDNA of human ODC cDNA and had 99% homology with human ODC cDNA of NIH gene library. The recombinant protein was transformed into Escherichia coli M 15 and the expressed protein was verified by Western Blotting as the target protein. Purified fusion protein, immunized mice to obtain multi-titers higher potency. Immunohistochemical detection of colorectal cancer ODC expression showed that the antibody test results better, colorectal cancer ODC expression was significantly higher than normal (P <0.05). Conclusion: The ODC expression vector was successfully constructed, ODC recombinant protein was expressed, and ODC polyclonal antibody was prepared, which laid a good foundation for further study on the pathogenesis of colorectal cancer and its diagnosis and treatment.