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目的 观察编码旋毛虫相对分子质量 (Mr) 3 10 0 0抗原的DNA疫苗 (重组真核表达质粒pcDNA3 TspE1)在中国仓鼠卵巢 (CHO)细胞中的体外表达 ,并分析其表达产物的抗原性。 方法 通过用阳离子脂质体Lipofectamine 2 0 0 0将重组质粒pcDNA3 TspE1转染CHO细胞 ,G418筛选阳性克隆 ,用逆转录 聚合酶链反应 (RT PCR)、间接荧光抗体试验 (I FAT)、十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白质印迹法 (Westernblotting)对表达产物进行鉴定。 结果 RT PCR结果显示 ,pcDNA3 TspE1转染的CHO细胞在 876bp处有一条带 ,而用空质粒pcDNA3转染的CHO细胞未出现条带 ,表明pcDNA3 TspE1转染细胞中有TspE1基因转录。IFAT结果显示 ,pcDNA3 TspE1转染的CHO细胞与重组融合蛋白免疫小鼠血清反应呈现亮绿色荧光 ,而pcDNA3转染的CHO细胞及未转染细胞呈现橘黄色。Westernblotting显示在pcDNA3 TspE1转染的CHO细胞培养液中存在有一Mr约 3 10 0 0的蛋白带 ,且该条带能被重组融合蛋白免疫小鼠血清、旋毛虫肌幼虫可溶性抗原免疫兔血清、感染旋毛虫的小鼠及旋毛虫病患者血清识别。 结论 重组质粒pcDNA3 TspE1可转染CHO细胞 ,旋毛虫TspE1基因可在转染的CHO细胞中表达 ,表达蛋白能分泌到细胞培养上清中且具有旋毛虫?
Objective To observe the in vitro expression of DNA vaccines (recombinant eukaryotic expression plasmid pcDNA3 TspE1) encoding Trichinella spiralis relative molecular weight (Mr3-1000) in Chinese hamster ovary (CHO) cells and analyze the antigenicity of the expressed product. Methods The CHO cells were transfected with the recombinant plasmid pcDNA3 TspE1 using Lipofectamine 2000. The positive clones were screened by G418. The positive clones were obtained by reverse transcription polymerase chain reaction (RT PCR), indirect fluorescent antibody test (I FAT) The expression products were identified by sodium polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Results RT-PCR showed that there was a band at 876bp in pcDNA3 TspE1-transfected CHO cells, but no band appeared in CHO cells transfected with empty plasmid pcDNA3, indicating that TspE1 gene was transcribed in pcDNA3 TspE1 transfected cells. The results of IFAT showed that the serum of pcDNA3 TspE1 transfected CHO cells and recombinant fusion protein showed bright green fluorescence, while the pcDNA3 transfected CHO cells and untransfected cells showed orange color. Western blotting showed that there was a protein band of Mr about 3 10 0 in pcDNA3 TspE1 transfected CHO cell culture medium and the band could be immunized with the recombinant fusion protein and the rabbit serum was immunized with soluble antigen of Trichinella spiralis larvae Trichinella mice and Trichinella patients serum identification. Conclusion The recombinant plasmid pcDNA3 TspE1 can be transfected into CHO cells. The TspE1 gene of Trichinella spiralis can be expressed in transfected CHO cells. The expressed protein can be secreted into the cell culture supernatant and has Trichinella spiralis.