Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma

Abstract

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.

Figure 1

RT-PCR product on 1% agarose gel. Lane 1: ODC RT-PCR product, 1480 bp in length; Lane 2: pQE30-ODC plas-mid digested by restriction enzyme BamH I and Sal I.

Figure 2

The pQE30-ODC expression vector including early promoter T5, Lac operator and 6His-Tag.

Figure 3

Expressed ODC protein on SDS-PAGE. Lane 1: M15 negative control; Lane 2-5: Expressed ODC proteins in M15 1 to 4 hour(s) after induction of IPTG; M: Protein marker; The arrow showed the expressed ODC protein.

Figure 4

Western blot shows a strip of 50 kD indicating the expression of ODC protein.