Construction of GAL’s Eukaryotic Expression Vector and Targeting Expression of GAL in N-2a Cells

Abstract

Abstract: The study was designed to construct eukaryotic expression vector pcDNA-PDGF-GAL, and observe the specific expression of GAL in N-2a after transferction. Firstly, the 2.9 kb PDGF-GAL fragment was amplified by PCR, then directedly cloned into pcDNA 3.1 vector, replacing the vector’s CMV promoter and enhancer and finally forming the recombinant vector pcDNA-PDGF-GAL which is neuronspecific expressed. Secondly, pcDNA-PDGF-GAL is transient-transfected into N-2a and the expression of GAL protein is detected by RT-PCR and ELISA. Results showed the sequencings result of pcDNA-PDGF-GAL was blasted on NCBI with standard sequence, in which PDGF-GAL fragment has a high nucleotide sequence identity（99%）, and these results suggested that the construction of recombinant plasmid gets success. By RT-PCR and ELISA, it is confirmed that GAL protein is specifically over-expressed in N-2a. The neuron-specific eukaryotic expression vector was successfully constructed, and GAL protein is successfully over-expressed in N-2a, which established a basis for the preparation of stabletransfected cells over-expressing GAL and further exploring the biological functions of GAL on nervous system.

Key words: GAL, Expression vector, N-2a cell, Transfection

Rong Man, Zhang Ruihu, Liu Tianfu . Construction of GAL’s Eukaryotic Expression Vector and Targeting Expression of GAL in N-2a Cells[J]. Biotechnology Bulletin, 2013, 0(2): 124-129.

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