Construction and identification of a mutated-Cdh1 eukaryotic expressing vector

doi:10.3969/j.issn.2095-4344.2013.02.023

Abstract

Abstract:

BACKGROUND: The activity of anaphase promoting complex-Cdh1 is regulated by phosphorylation.
Phosphorylated Cdh1 cannot be combined with anaphase promoting complex, thereby inhibiting the activity
of the anaphase promoting complex.
OBJECTIVE: To construct and identify a mutated-Cdh1 eukaryotic expressing vector.
METHODS: The entire coding sequence of the Cdh1 gene was amplified from rat hippocampal mRNA by reverse transcription-PCR. Then the PCR product of Cdh1 was cloned into pBluescript plasmid by double digestion with restriction endonucleases EcoR1 and Xba1, and ligation. Based on site-directed mutagenesis, the pBluescript-Cdh1 plasmid containing Cdh1 coding sequence was used as a template. The 40, 151, 163 serine (S) and 121 threonine (T) in Cdh1 gene was mutated to alanine (A) by multiple PCR with four pairs of mutated primers. The mutated Cdh1 vector was identified by DNA sequencing.
RESULTS AND CONCLUSION: The PCR product was about 1 500 bp by electrophoresis, including the entire coding sequence of Cdh1, restriction sites at both ends of Cdh1 and KOZAK sequence. The recombinant pBluescript-Cdh1 plasmid was identified by digestion with restriction endonuclease EcoR1 and Xba1, which was consistent with the expected results. DNA sequencing showed that A at the 930th base of Cdh1 (BC162059.1) coding sequence was mutated to G in the recombinant plasmid of pBluescript-Cdh1. But the sequence of amino acids was not affected. The 40, 121, 151, 163 amino acids in pBluescript-Cdh1-4A40, 121, 151, 163 mutant plasmids were all mutated to alanine. The mutated Cdh1 gene expressing plasmid at phosphorylation site was successfully constructed, which provides a good foundation for further studies of Cdh1 gene function.

Key words: tissue construction, cytological experiments of tissue construction, anaphase promoting complex, Cdh1, site-directed mutagenesis, PCR, eukaryotic expression plasmid, nervous system, development, nerve repair, gene therapy, National Natural Science Foundation of China

CLC Number:

R318

Li Li, Shi Xiao-yun, Zhang Deng-wen, Zhang Chuan-han, Yao Wen-long. Construction and identification of a mutated-Cdh1 eukaryotic expressing vector[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(2): 315-319.

Figures/Tables 4

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