Aim
To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma.
Conclusion
The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in 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.