旋毛虫DNA疫苗在中国仓鼠卵巢细胞中的表达(英文)

Expression of DNA Vaccine against Trichinella spiralis in Chinese Hamster Ovary Cells

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 vaccine (recombinant eukaryotic expression plasmid pcDNA3 TspE1) encoding a Mr 31 000 antigen of Trichinella spiralis in Chinese hamster ovary (CHO) cells and analyze the antigenicity of the products expressed. Methods The recombinant plasmid pcDNA3 TspE1 was transfected into CHO cells by using cationic lipids (Lipofectamine 2000). The positive cell clones were screened by the selective antibiotic G418. The expressed products were identified by RT PCR, IFAT, SDS PAGE and Western blotting. Results The results of RT PCR amplification showed that there was one band with 876 bp in CHO cells transfected with pcDNA3 TspE1 and no any bands in CHO cells transfected with the empty plasmid pcDNA3. The IFAT demonstrated that the pcDNA3 TspE1 transfected CHO cells reacted with sera from mice immunized with the recombinant fusion protein and from mice infected with T. spiralis , the bright yellow green fluorescence staining appeared in the transfected CHO cells. The pcDNA3 transfected and un transfected CHO cells exhibited as orange color. The results of SDS PAGE showed that there was one band with Mr 31 000 in culture supernatant of CHO cells transfected with pcDNA3 TspE1. Western blotting confirmed that the band with Mr 31 000 could be recognized by sera from mice immunized with the recombinant fusion protein, from rabbits immunized with T. spiralis muscle larval soluble antigens, from mice infected with T. spiralis and from patients with trichinellosis. Conclusion The mammalian CHO cells were transfected by the recombinant plasmid pcDNA3 TspE1, and the TspE1 gene of T. spiralis was expressed in the transfected CHO cells. The proteins expressed are secreted into cell culture supernatants and show the antigenicity of T. spiralis.