鼠伯氏疟原虫CSP基因真核表达载体的构建及在Hela细胞中的表达

Construction of A CSP (Circumsporozoite Protein) Eukaryotic Expression Vector from Plasmodium berghei

目的分别构建携带绿色荧光蛋白报告基因的伯氏疟原虫(Plasmodiumberghei)环子孢子(CSP)全长基因和去除中央重复序列的CSP的融合蛋白真核表达质粒,并检测其在Hela细胞中的表达。方法采用PCR技术,从伯氏疟原虫基因组中扩增全长CSP基因,将其定向克隆入pEGFP-N1,构建pEGFP/PbCSP全长基因重组质粒,酶切、PCR及序列分析鉴定;同样,采用PCR技术扩增PbCSP的N端和C端两个片段,先将C端片段定向克隆入pEGFP-N1,构建pEGFP/PbCSP-C重组质粒,再将PbCSPN端片段定向克隆入pEGFP/PbCSP-C,构建pEGFP/PbCSP'重组质粒,酶切、PCR及序列分析鉴定。将pEGFP/PbCSP和pEGFP/PbCSP′分别用脂质体介导转染体外培养的Hela细胞,荧光显微镜及RT-PCR检测融合蛋白的表达。结果PCR、酶切及测序证实目的基因PbCSP全长和片段分别正确连接至pEGFP-N1的多克隆位点,两种重组质粒转染Hela后,荧光显微镜及RT-PCR均检测到目的蛋白在Hela中表达。结论成功构建了携带绿色荧光蛋白报告基因的伯氏疟原虫CSP全长基因和该基因片段的融合蛋白真核表达质粒,并在Hela细胞中获得表达。

Objective To construct the CSP （ciroumsporozoite protein） expression vector pEGFP-N1 and to express the fusion green fluorescent protein PbCSP/pEGFP in HeLa cells. Method The PbCSP gene was obtained from the genomic DNA of P.berghei by PCR amplification. The PCR products were cloned into pEGFP-N1 vector and the construct was confirmed by PCR and restriction enzyme digestion. The pEGFP/PbCSP-C construct was obtained by cloning the C-terminal fragment of PbCSP into pEGFP-N1. The N-terminal of PbCSP was then cloned into pEGFP-N1/PbCSP-C to produce the construct pEGFP/PbCSP′. Transient expression of the fusion protein was observed at 48 hours after transfection with pEGFP/PbCSP and pEGFP/PbCSP′. Result A full length eukaryotic expression vector pEGFP/PbCSP was constructed. Conclusion pEGFP/PbCSP and pEGFP/PbCSP′ can be expressed in HeLa cells.