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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 eukaryotic expression plasmids of Plasmodium berghei circumsporozoite (CSP) full length gene carrying green fluorescent protein reporter gene and CSP with central repeats, and to detect the expression of the fusion protein in Hela cells . Methods The full-length CSP gene was amplified from the genome of Plasmodium berghei by PCR and cloned into pEGFP-N1. The full-length recombinant plasmid pEGFP / PbCSP was constructed and identified by restriction enzyme digestion, PCR and sequence analysis. Similarly, PCR technique was used to amplify the N-terminal and C-terminal fragments of PbCSP. The C-terminal fragment was cloned into pEGFP-N1 first and then the recombinant plasmid pEGFP / PbCSP-C was constructed. The fragment of PbCSPN was cloned into pEGFP / PbCSP- Construction of pEGFP / PbCSP recombinant plasmid, digestion, PCR and sequence analysis. The pEGFP / PbCSP and pEGFP / PbCSP ’were transfected into Hela cells by lipofectamine respectively. The expression of fusion protein was detected by fluorescence microscopy and RT-PCR. Results PCR, restriction enzyme digestion and sequencing confirmed that the target gene PbCSP full length and fragment were correctly linked to the pEGFP-N1 multiple cloning site, two recombinant plasmids transfected Hela, the fluorescence microscope and RT-PCR detected the target protein in Hela In the expression. Conclusion The full-length CSP gene of P. berghei carrying green fluorescent protein reporter gene and the fusion protein eukaryotic expression plasmid of this gene fragment were successfully constructed and expressed in Hela cells.