人结直肠癌鸟氨酸脱羧酶基因的克隆与表达
Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma.
作者信息
Hu Hai-Yan, Liu Xian-Xi, Jiang Chun-Ying, Zhang Yan, Bian Ji-Feng, Lu Yi, Geng Zhao, Liu Shi-Lian, Liu Chuan-Hua, Wang Xiao-Ming, Wang Wei
机构信息
Experimental Centre of Medical Molecular Biology, School of Medicine, Shandon University, Jinan 250012, Shandong Province, China.
出版信息
World J Gastroenterol. 2003 Apr;9(4):714-6. doi: 10.3748/wjg.v9.i4.714.
AIM
To construct and express ODC recombinant gene for further exploring its potential use 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 facilitated for purification by Ni-NTA chromatographic column.
RESULTS
ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E. coli M15 and expressed. The expressed ODC protein was verified with Western blotting.
CONCLUSION
The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.
目的
构建并表达鸟氨酸脱羧酶（ODC）重组基因，以进一步探索其在结直肠癌早期诊断中的潜在应用。
方法
从结肠癌组织中提取总RNA，使用跨越ODC整个编码区的两条引物通过逆转录聚合酶链反应（RT-PCR）进行扩增。将合成的ODC cDNA在BamH I和Sal I限制性酶切位点克隆到载体pQE-30中，构建重组表达质粒pQE30-ODC。通过DNA测序确认插入片段的序列，含6His标签的融合蛋白便于用镍-氮三乙酸（Ni-NTA）层析柱进行纯化。
结果
构建了ODC表达载体，并通过限制性酶切和后续DNA测序进行了确认。在NCBI Blast上的DNA序列比对显示亲和力为99%。将该载体转化到大肠杆菌M15中并进行表达。通过蛋白质免疫印迹法（Western blotting）验证了表达的ODC蛋白。
结论
构建了ODC原核表达载体，极大地有助于研究ODC在结直肠癌中的作用。
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