Construction of eukaryotic expression vectors of two NF2 mutants and their expression in rat schwannoma cell line
10.3724/SP.J.1008.2008.00546
- Author:
Hua HE
1
Author Information
1. Department of Neurosurgery
- Publication Type:Journal Article
- Keywords:
Eukaryotic expression vector;
NF2;
Point mutation
- From:
Academic Journal of Second Military Medical University
2010;29(5):546-549
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct the eukaryotic expression vectors of two NF2 mutants (neurofibromatosis II) and to study their expression in rat schwannoma cell line (RT4). Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Lys42 in NF2 into Pro in pcDNA3. 1-NF2ΔLys42Pro, and convert the codons for Phe 47 into that of Leu. RT4 cells were transiently transfected with the 3 kinds of plasmids containing the mutations and wide-type NF2 via lipofectin separately, then the expression levels of NF2 mRNA and protein were determined by RT-PCR and Western blotting in 3 groups. The cell proliferation was determined by the MTT after transfection. Results: DNA sequence analysis demonstrated the two-step mutagenesis was successful and the two plasmids of pcDNA3.1ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu were obtained, both of the transfectants could produce merlin protein and mRNA. The two mutants had a significantly lower inhibitory rate for RT4 cells compared with wide-type NF2 (P < 0.05). Conclusion: The recombinant plasmids pcDNA3. 1-NF2ΔLys42Pro and pcDNA3. 1-NF2ΔPhe47Leu have been successfully constructed and they can be efficiently expressed in RT4 cells.
