Construction and expression of the eukaryotic expressed plasmid of gene from in IBRS-2 cells

Abstract

The sequence encoding MIC3 was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the vector pMD18-T. The target gene was subcloned into the eukaryotic vector pcDNA3.1 after the identification of pMD18-T-MIC3 by enzyme digesting, PCR amplification and sequencing. Then the target recombinant plasmids pcMIC3 were transfected into IBRS-2 cells, and the positive cells containing pcMIC3 plasmids were obtained under the selection of G418. The expressed proteins from the positive cells were detected by SDS-PAGE, Western blot and ELISA. The results showed that the DNA sequence identity was 99.9% between amplified MIC3 and that from GenBank. The molecular weight of the recombinant MIC3 protein with good immuno-activity was about 39.2 ku. These available data would lay the foundation for further studies on DNA vaccine against Toxoplasma gondii.

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