Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene |
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Authors: | Shengjun Ren Huiqiu Jiang Jianren Gu |
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Institution: | (1) National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, 200032 Shanghai, China |
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Abstract: | Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression
vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine
deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence
analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide
by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank. |
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Keywords: | cytosine deaminase gene prokaryotic expression vector SDS-PAGE analysis cytosine deaminase activity assay high activity clone of CD |
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