Aim: To construct the eukaryotic recombinant expression plasmid of pcDNA3.1(+)-PRMT1.
Methods: Human PRMT1 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). After digested by BamH I, Hind III and ligation, PRMT1 was inserted into pcDNA3.1(+)eukaryotic expression vector. The positive colonies were screened and identified by PCR and sequencing. pcDNA3.1(+)-PRMT1 plasmid was then transfected into the cultured A549 cell line with Lipofectamine(TM);2000. Realtime-PCR and Western blot were used to detect the mRNA and protein expression of PRMT1 respectively.
Results: The PRMT1 cDNA was successfully amplified, and pcDNA3.1(+)-PRMT1 were constructed. The inserted sequence in pcDNA3.1(+)-PRMT1 was the same as the sequence of PRMT1 cDNA published in NCBI GenBank. Further, Realtime PCR and Western blot results validated the recombinant plasmid expressed in A549 cell line efficiently.
Conclusion: pcDNA3.1(+)-PRMT1 recombinant was successfully constructed.