Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma.
OBJECTIVE
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.


CONCLUSIONS
The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.
Hu
Hai-Yan
HY
Experimental Centre of Medical Molecular Biology, School of Medicine, Shandon University, Jinan 250012, Shandong Province, China.
Liu
Xian-Xi
XX
Jiang
Chun-Ying
CY
Zhang
Yan
Y
Bian
Ji-Feng
JF
Lu
Yi
Y
Geng
Zhao
Z
Liu
Shi-Lian
SL
Liu
Chuan-Hua
CH
Wang
Xiao-Ming
XM
Wang
Wei
W
D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000601:surgery; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D009955:Ornithine Decarboxylase / Q000235:genetics*; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009955:Ornithine Decarboxylase