Objective To clone S100A4 cDNA and construct pcDNA3.1-S100A4 recombinant expression vector, transfect gastric cancer cell line MKN1, and observe the expression of S100A4 in gastric cancer cells.
Methods Total RNA of human gastric epithelial cells GES-1 was extracted by Trizol method, and cDNA containing S100A4 gene was obtained by reverse transcription reaction. The sequence of S100A4 gene was obtained from GenBank by polymerase chain reaction (PCR), and primers were designed to amplify the product with a molecular weight of 327 bp. The pMD18-T simple-S100A4 recombinant plasmid was constructed to transform JM109 bacteria. After the bacterial solution was sequenced successfully, the plasmid was extracted for double enzyme digestion by BamH Ⅰ/Hind Ⅲ, and the enzyme digestion products were recovered and purified, and the expression vector pcDNA3.1 was connected to construct PCDNA3.1-S100A4 eukaryotic expression vector. The purified expression vector was transfected into gastric cancer cell line MKN1 by liposome mediated transfection method. The expression level of S100A4 mRNA in transfected MKN1 cells was detected by reverse transcription polymerase chain reaction (RT-PCR).
Results The sequencing results of the cloned vector linked with PCR products were consistent with the mutation-free sequence published by GenBank, and the eukaryotic expression vector pcDNA3.1-S100A4 could be successfully constructed by double enzyme digestion of Hind Ⅲ/BamH Ⅰ; pcDNA3.1-S100A4 expression vector was transfected into gastric cancer cell line MKN1, and transfected empty pcDNA3.1 vector and normal untransfected MKN1 cells were used as controls. The results showed that the expression level of S100A4 mRNA was increased 48 h after transfection with pcDNA3.1-S100A4 expression vector (P < 0.01).
Conclusion The eukaryotic expression vector pcDNA3.1-S100A4 is constructed, and S100A4 gene is successfully expressed in gastric cancer cell MKN1.
DownLoad: