日本血吸虫真核表达重组质粒pcDNA3-Sj26的构建及其在树突状细胞中的表达

CONSTRUCTION OF EUKARYOTIC EXPRESSION RECOMBINANT PLASMID PCDNA3-SJ26 OF SCHISTOSOMA JAPONICUM AND ITS EXPRESSION IN DENDRITIC CELL

本研究构建日本血吸虫2 6kDa谷胱苷肽转移酶(Sj2 6 )基因的真核表达重组质粒(pcDNA3 Sj2 6 ) ,并探讨其在树突状细胞(DC)中的表达。采用PCR扩增Sj2 6基因片段,定向克隆至真核表达载体pcDNA3中,阳性克隆经双酶切、PCR扩增鉴定后,双脱氧链末端终止法进行序列测定。用脂质体介导DNA转染法将pcDNA3- Sj2 6转染DC ,G4 18加压筛选获得阳性克隆并传代培养,用RT PCR检测Sj2 6mRNA的转录水平,用SDS- PAGE、Westernblot和间接免疫荧光法检测Sj2 6蛋白在DC中的表达。结果PCR扩增得到特异的Sj2 6基因片段,双酶切及PCR鉴定表明获得正确的pcDNA3 -Sj2 6重组质粒;测序结果表明,核苷酸序列和推导的氨基酸序列与GenBank报道的Sj2 6的一致性均为99% ;RT PCR结果显示pcDNA3- Sj2 6转染的DC中有Sj2 6基因mRNA的表达,SDS -PAGE和Westernblot、间接免疫荧光法显示pcDNA3- Sj2 6转染的DC中有Sj2 6的表达。结果证实构建的真核表达重组质粒pcDNA3- Sj2 6成功的转染至DC中并在DC中获得表达。

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 DH5α.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 SDS-PAGE,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.