Molecular cloning and functional expression of protein kinase C μ in Chinese hamster ovary cell
Xie Wen-li · Acta Academiae Medicinae Cpapf · 2007
【Objective】To study the expression of PKC μ in Chinese hamster ovary cell(CHO cell).【Methods】The PKC μ primer pairs were derived from GeneBank sequence information based on similar species sequence.Using EMBL Data Library CLUSTALW tool,the highest homologues sequences were screened among the different species for PKC μ,and were used to design the primers.The primers were designed by using Generunner program.PKC μ was profiled by the RT-PCR technique.PKC μ was ligated to the pGEM-T plasmid vector.Transformants were taken from E.coli DH5α.Blue/white screening for recombinants in indicator plate.The plasmid construct was digested with restriction enzyme and was analyzed with gel electrophoresis.The product of restriction digestion with the sizes consistent and the predicated values was sent for DNA sequencing.Western blotting was used to analyze the PKC μ expressed in CHO cell.【Results】The presence of mRNA for the PKC μ expressed in CHO cell was determined by RT-PCR.PKC μ was found in CHO cell by Western blot analysis.【Conclusion】PKC μ was in CHO cell.That lays solid basis for further study the structure and function of PKC μ directly and the relationship with signal transduction pathways in cell.