Guo Ling-ling, Zhang Xiao-lei, Zhang Jin-shun, Jia Xiao-hui, Wang Chun-miao, Jiang Wen-jing, Zhu Xiao-bo, Jia Tian-jun
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2015 Jun;33(3):161-6.
To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells.
Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I . ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene β-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein.
Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind III, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for β-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2,373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12.
The recombinant eukaryotic plasmid pVAX1-ROP18-ROP-2 is constructed and can be expressed in eukaryotic system.
构建含弓形虫棒状体蛋白18-12（ROP18-ROP12）复合基因的重组真核表达质粒，并检测其在真核细胞中的表达。
用限制性内切酶BamHⅠ和XbaⅠ分别酶切重组质粒pVAX1-ROP18和pVAX1-ROP12。将ROP12基因克隆至pVAX1-ROP18中构建真核表达质粒pVAX1-ROP18-ROP12。经菌落PCR、酶切及测序鉴定后，将正确的重组质粒pVAX1-ROP18-ROP12转染入HeLa细胞。同时设置空质粒组、pVAX1-ROP18组和pVAX1-ROP12组。提取HeLa细胞的总RNA并反转录为cDNA。采用RT-PCR检测管家基因β-肌动蛋白及ROP18-ROP12复合基因的mRNA表达。采用免疫荧光法和蛋白质印迹法检测ROP18-ROP12融合蛋白的表达水平。
重组质粒pVAX1-ROP18-ROP12的菌落PCR显示在约2373 bp处出现特异性条带，与预期相符。提取的重组质粒经HindⅢ、BamHⅠ和XbaⅠ酶切鉴定。测序结果显示，pVAX1-ROP18-ROP12序列与弓形虫RH株ROP18基因（登录号：AM075204.1）100%相同，与弓形虫RH株ROP12基因（登录号：DQ096559.1）99%相同。此外，RT-PCR结果显示，所有组β-肌动蛋白均在613 bp处出现扩增产物，而只有pVAX1-ROP18-ROP12转染组在2373 bp处出现ROP18-ROP12复合基因的扩增产物。间接免疫荧光法显示，pVAX1-ROP18-ROP12转染的HeLa细胞呈现黄绿色荧光，而对照细胞未出现。蛋白质印迹法显示，重组质粒pVAX1-ROP18-ROP12转染的HeLa细胞中有ROP18-ROP12融合蛋白表达。
成功构建了重组真核质粒pVAX1-ROP18-ROP12，且能在真核系统中表达。
Zotero 插件