重组质粒pcDNA3-gtfB在哺乳动物细胞中的表达

Expression of Plasmid pcDNA3-gtfB in Mammalian Cell

目的 :检测含有变形链球菌葡糖基转移酶抗原基因 (gtfB)的重组质粒pcDNA3_gtfB能否在哺乳动物细胞COS_1中正确地转录和表达。方法 :采用脂质体介导法 ,将pcDNA3_gtfB转染哺乳动物细胞COS_1,采用RT_PCR法、免疫组化LSAB法、Western免疫印迹法检测重组质粒的转录水平和表达产物。结果 :pcDNA3_gtfB在转染COS_1细胞后具有转录和翻译活性 ,蛋白质表达产物分子量为 116～ 2 12kD ,且可与兔抗葡糖基转移酶抗体发生特异性结合。结论 :重组质粒pcDNA3_gtfB在哺乳动物细胞中能正确地转录和表达 。

Objective:This study aimed at investigating the transcription and expression of recombined plasmid pcDNA3-gtfB which encoding multiple glucosyltransferase-B antigenic gene, and the feasibility of the pcDNA3-gtfB used as gene vaccine.Methods: The pcDNA3-gtfB was transfected into mammalian cell COS-1 with liposome. The total RNA of COS-1 cell transfected by pcDNA3-gtfB was extracted and purified. Using the total RNA as template, the transcription of pcDNA3-gtfB was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of pcDNA3-gtfB was identified with 5% SDS-PAGE, and then assayed using Western-blotting. The expression product of pcDNA3-gtfB was also assayed by using LSAB method, and cell transfected by pcDNA3 as the negative control.Results: Identified by agarose gel electrophoresis, the target gene fragment had the same molecular size (3.6 kb) as it was predicted, and it indicated that pcDNA-3gtfB was correctly transcribed into mammalian cells. Proved by SDS-PAGE, the molecular weight of the expression product (116～212 kD) was also the same as it was supposed to be. It was also indicated by Western-blotting and LSAB assay that the expression product induced immunizing response.Conclusion: As gene vaccine, it is importance that the recombined plasmid could be correctly transcribed and expressed in mammalian cells. It was suggested by RT-PCR, LSAB and Western-blotting that recombined plasmid pcDNA3-gtfB could be correctly transcribed and expressed in mammalian cells, and the expression product could induce immunizing response, which support its use as gene vaccine candidates in the development of anticaries vaccines.