摘要
为了提高猪睾丸(swine testicular,ST)细胞的转染效率,研究猪传染性胃肠炎病毒(Porcine transmissible gastroenteritis virus,TGEV)各蛋白的功能,试验利用绿色荧光蛋白(green fluorescent protein,GFP)的真核表达载体pEGFP-C1转染ST细胞,对ST细胞的转染条件进行优化;然后利用优化后的转染条件将TGEV各蛋白真核表达质粒转染到ST细胞,通过免疫印迹(Western-blot)和间接免疫荧光(IFA)方法检测TGEV各蛋白在ST细胞中的表达情况。结果表明:悬浮细胞转染法(在细胞传代的同时进行转染)比贴壁细胞转染法(待细胞贴壁后再进行转染)的转染效率更高,且转染质粒与转染试剂的比例为1∶3时是转染ST细胞的最佳比例。利用优化后的转染条件将TGEV各蛋白真核表达质粒转染到ST细胞,48 h后采用IFA法检测发现TGEV各蛋白在ST细胞中成功表达,Western-blot法检测发现TGEV各蛋白在ST细胞中表达的大小与预期相符。说明优化后的转染条件可用于提高ST细胞的转染效率。
The aim of the present study was to improve the transfection efficiency of swine testicular ( ST) cells and further study the function of the proteins of porcine transmissible gastroenteritis virus ( TGEV) . The eukaryotic expression vector pEGFP -C1 expressing green fluorescent protein ( GFP) was used to transfect ST cells,and the transfection conditions of ST cells were optimized. Then the eukaryotic expression plasmids expressing each TGEV proteins were transfected into ST cells under the optimized transfection conditions. The expression of TGEV proteins in ST cells was detected by Western-blot and indirect immunofluorescence ( IFA) . The results showed that the transfection efficiency of suspension cell transfection ( the transfection was carried out in the passage of the cells) was higher than that of adherent cell transfection ( the transfection was carried out after the cell adheres to the wall) . When the ratio of transfection plasmid to transfection reagent was 1: 3,the transfection efficiency of ST cells was the best. The eukaryotic expression plasmids expressing TGEV proteins were transfected into ST cells under the optimized transfection conditions. After 48 hours,all TGEV proteins were successfully expressed in ST cells by IFA assay. The expression of TGEV proteins in ST cells was in accordance with the expected size by Western-blot method assay. The results indicated that the optimized transfection conditions can be used to improve the transfection efficiency of ST cells.
作者
王丽
郭田田
秦越
高玉
孙刘妹
李海滨
李一经
WANG Li;GUO Tiantian;QIN Yue;GAO Yu;SUN Liumei;LI Haibin;LI Yijing(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2018年第23期17-20,265,266,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
黑龙江省教育厅科学技术研究项目(12541031)
关键词
猪传染性胃肠炎病毒
猪睾丸细胞
真核表达
转染
免疫印迹
间接免疫荧光
porcine transmissible gastroenteritis virus
swine testicular cells
eukaryotic expression
transfection
Western-blot
indirect immunofluorescence
