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Construction of eukaryotic expression recombinant plasmid PCDNA-3-SJ26 of Schistosoma japonicum and its expression in dendritic cell


Construction of eukaryotic expression recombinant plasmid PCDNA-3-SJ26 of Schistosoma japonicum and its expression in dendritic cell



Jishengchong Yu Yixue Kunchong Xuebao ; 12(2): 65-70



This study aimed to construct the eukaryotic expression recombinant plasmid of Schistosoma japonicum (pcDNA3-Sj26) and to explore its expression in dendritic cell (DC) in vitro. Sj26 gene was amplified from the plasmid pGEX-3X by PCR and cloned into the eukaryotic expression vector pcDNA3. Recombinant plasmid pcDNA3-Sj26 was constructed and transferred into E. coli DHSa. The positive clones were screened and identified by endonuclease digestion and PCR amplification. The nucleotide sequence of Sj26 gene was determined by the method of dideoxy chain termination. Recombinant plasmid pcDNA3-Sj26 was introduced into DC by liposome-mediated gene transfer method. The positive DC clones were obtained using G418 selected-screening approach. RT-PCR was used to analysis the expression of Sj26 mRNA and the expression of Sj26 protein were determined by SDSPAGE, Western blot and indirect immunofluorescence techniques. Sj26 gene was specifically amplified. The correct recombinant plasmid pcDNA-Sj26 was constructed. The identities of both nucleotide acid and deduced amino acid sequence of Sj26 gene were 99% , compared to that of Sj26 gene in the GenBank. Using RT-PCR method, the expression of Sj26 mRNA was confirmed in the pcDNA3-Sj26 transfer DC. The results of SDS-PAGE, Western blot and indirect immunofluorescence method showed that there was Sj26 protein expressed in the pcDNA3-Sj26 transfer DC, which could be specifically recognized by infective mouse serum. The eukaryotic expression recombinant plasmid pcDNA3-Sj26 was successfully constructed and could be transfected and expressed in DC in vitro.

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