摘要
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.
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.
基金
Scientific Research Fund of national Ministry of Health,No.98-1-173
