刚地弓形虫RH株ROP18基因真核表达载体的构建

Construction and expression of a eukaryotic expression vector containing the rhoptry protein 18(ROP18)gene of the RH strain of Toxoplasma gondii

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。

Objectives To clone the rhoptry protein 18 （ROP18） gene of To：coplasrna gondii and to construct the re- combinant plasmid pVAX1-ROP18 and examine its expression in eukaryotic cells. Methods The ROP18 gene and the plasmid pVAX1 were digested with the restriction enzymes Hind III and BamH I, respectively. After ligation overnight with T4 ligase, the products of ligation were transformed into 17.. coil XL1-Blue competent cells. The recombinant plas- mid pVAX1-ROP18 was verified to be correct with colony PCR, double enzyme digestion, and sequencing. The recombi- nant plasmid was then transfected into HeLa cells. An empty plasmid group and a blank group were also created. Total RNA was extracted from HeLa cells in each group and was reverse transcribed into cDNA. β-actin and ROP18 were am- plified with RT-PCR to determine whether the ROP18 gene was transcribed. Expression of ROP18 protein was observed with Western blotting. Results After agarose gel electrophoresis, RT-PCR yielded a specific fragment of about 1,665 bp in length, which was consistent with expectations. Colony PCR yielded a product of 1,665 bp in length, which was consistent with expectations. The recombinant plasmid pVAX1-ROP18 was correct according to double digestion. Se- quencing of the ROP18 gene revealed that it was consistent with the ROP18 gene from the RH strain of To：coplaszna gon- dii in GenBank （Accession No. AM075204.1. After liposomal transfection, RT-PCR of β-actin from each group yielded a product 613 bp in length, which was consistent with expectations. RT-PCR of the ROP18 gene in cells transfected with the recombinant plasmid pVAXI-ROP18 yielded a product 1,665 bp in length, which was consistent with expectations. A similar target band was not noted in other cells. Western blotting indicated that the recombinant plasmid pVAXI-ROP18 expressed ROP18 protein in HeLa cells. Conclusions The recombinant plasmid pVAXI-ROP18 was successfully con- structed and it expressed ROP18 protein in eukaryotic cells.