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目的构建刚地弓形虫棒状体蛋白16(TgROP16)、棒状体蛋白18(TgROP18)复合基因真核表达重组质粒,并验证其在真核细胞中的表达情况。方法重组质粒pVAX1-ROP16与pVAX1-ROP18分别经EcoRⅠ和NotⅠ、NheⅠ和AflⅡ双酶切,将ROP16与ROP18基因先后克隆至双启动子真核表达载体质粒pVAXD的多克隆位点中,构建pVAXD-ROP16-ROP18重组质粒。分别用双酶切和PCR验证正确的重组质粒及对照组质粒转染Hela细胞,提取细胞总RNA并逆转录为cDNA,以此为模板进行ROP16基因、ROP18基因与管家基因β肌动蛋白基因的RT-PCR扩增,采用间接免疫荧光法检测各自基因的表达。结果重组质粒pVAXD-ROP16-ROP18经PCR及双酶切鉴定构建正确。转染后经RT-PCR检测,实验组与对照组β-肌动蛋白基因扩增产物均与预期大小相符,实验组ROP16基因、ROP18基因扩增产物分别为1 700bp和2 100bp,与预期大小相符,而对照组均未扩增出ROP16及ROP18基因。间接免疫荧光法检测显示,重组质粒pVAXD-ROP16、pVAXD-ROP18转染组细胞可见绿色荧光,空质粒及对照组无绿色荧光。结论成功构建了重组质粒pVAXD-ROP16-ROP18,该质粒可在真核细胞中表达。
Objective To construct eukaryotic recombinant plasmids of TgROP16 and TgROP18 and to verify their expression in eukaryotic cells. METHODS: The recombinant plasmids pVAX1-ROP16 and pVAX1-ROP18 were digested with EcoRⅠ, NotⅠ, NheI and AflⅡ, respectively. The ROP16 and ROP18 genes were cloned into the multi-cloning site of the eukaryotic expression plasmid pVAXD. ROP16-ROP18 recombinant plasmid. Restriction endonuclease digestion and PCR were used to verify the correct recombinant plasmids and control plasmids were transfected into Hela cells, total cellular RNA was extracted and reverse transcribed into cDNA. The ROP16 gene, ROP18 gene and housekeeping gene β-actin gene RT-PCR amplification, indirect immunofluorescence assay of the respective gene expression. Results The recombinant plasmid pVAXD-ROP16-ROP18 was identified by PCR and restriction enzyme digestion. The results of RT-PCR showed that the amplification products of β-actin gene in experimental group and control group were consistent with the expected size. The amplified products of ROP16 gene and ROP18 gene in experimental group were 1 700bp and 2 100bp, respectively, However, ROP16 and ROP18 genes were not amplified in the control group. Indirect immunofluorescence assay showed that green fluorescence was observed in cells transfected with pVAXD-ROP16 and pVAXD-ROP18, and no green fluorescence was observed in empty plasmid and control cells. Conclusion The recombinant plasmid pVAXD-ROP16-ROP18 was successfully constructed and expressed in eukaryotic cells.