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目的 PCR扩增刚地弓形虫(Toxplasma gondii)棒状体蛋白18(ROP18)基因,构建真核表达载体pVAX1-ROP18,并检验其在真核细胞中的表达。方法 ROP18基因和质粒pVAX1分别经HindⅢ和BamHⅠ双酶切,在T4连接酶作用下连接过夜,将连接产物转化至大肠埃希菌E.coil XL1-Blue感受态细胞,经菌落PCR、双酶切及测序正确的重组质粒pVAX1-ROP18转染至HeLa细胞内。试验设空白对照组和pVAX1空质粒对照。提取各组HeLa细胞的总RNA并将其逆转录成cDNA,分别进行管家基因β-actin和ROP18基因的RT-PCR扩增;采用Western blot法检测转染后各组细胞ROP18蛋白的表达情况。结果经0.8%琼脂糖凝胶电泳检测,ROP18基因的RT-PCR扩增片段大小为1 665bp,与预期值相符;ROP18基因重组质粒转化菌菌落PCR产物经0.8%琼脂糖凝胶电泳鉴定,大小为1 665bp,与预期值相符;双酶切重组质粒得到预期酶切片段;重组质粒的目的基因序列与弓形虫RH株ROP18基因(登录号AM075204.1)序列比对完全一致。脂质体转染后各组β-actin的RT-PCR扩增目的片段均为613bp,与预期值相符;pVAX1-ROP18重组质粒转染组的ROP18的RT-PCR扩增目的片段大小为1 665bp,而其他组无该目的基因条带。Western blot检测重组质粒pVAX1-ROP18能够在HeLa细胞内表达ROP18蛋白。结论成功构建了真核表达载体pVAX1-ROP18,且该重组质粒能够在真核细胞内表达ROP18蛋白。
Objective To amplify the gene ROP18 of Toxoplasma gondii and construct eukaryotic expression vector pVAX1-ROP18, and to test its expression in eukaryotic cells. METHODS: ROP18 gene and plasmid pVAX1 were double digested with Hind Ⅲ and BamH Ⅰ, respectively, and ligated overnight with T4 ligase. The recombinant product was transformed into Escherichia coli E. coli XL1-Blue competent cells. After colony PCR and double enzyme digestion And the correct sequencing of the recombinant plasmid pVAX1-ROP18 transfected HeLa cells. The experiment set blank control group and pVAX1 empty plasmid control. The total RNA of HeLa cells in each group was extracted and reverse transcribed into cDNA. RT-PCR amplification of housekeeping genes β-actin and ROP18 respectively was performed. The expression of ROP18 protein in each group was detected by Western blot. Results The 0.875 agarose gel electrophoresis showed that the size of ROP18 gene amplified by RT-PCR was 1 665bp, which was consistent with the expected value. The PCR products of ROP18 recombinant plasmid transformed bacteria were identified by 0.8% agarose gel electrophoresis, Was 1 665bp, which was in accordance with the expected value. The recombinant plasmid was digested with the restriction enzyme to obtain the expected fragment. The sequence of the target gene of the recombinant plasmid was exactly the same as the sequence of ROP18 gene (Accession No. AM075204.1). RT-PCR amplification of β-actin in each group after transfection was 613bp, which was consistent with the expected value; the size of the target fragment of RT-PCR amplification of ROP18 in pVAX1-ROP18 transfection group was 1 665bp , While other groups did not have the target gene band. The recombinant plasmid pVAX1-ROP18 was able to express ROP18 protein in HeLa cells by Western blot. Conclusion The eukaryotic expression vector pVAX1-ROP18 was successfully constructed and the recombinant plasmid can express ROP18 protein in eukaryotic cells.