牙龈卟啉菌牙龈蛋白酶K基因真核表达载体的构建和体外表达

Construction of eukaryotic expression vector for KGPcd gene and its expression in vitro

目的 :构建牙龈卟啉菌牙龈蛋白酶K基因的真核表达载体 ,并检测其在体外真核细胞中的表达情况。方法 :利用基因重组技术构建牙龈卟啉菌牙龈蛋白酶K基因重组质粒pcDNA3.1( + ) -KGPcd ,然后通过脂质体将 pcDNA3.1( + ) -KGPcd瞬时转染COS7细胞 ,以RT -PCR和间接免疫荧光法检测重组质粒的转录水平和蛋白表达情况。结果 :成功构建了牙龈蛋白酶K真核表达载体 ;转染的COS7细胞中可检测到目的蛋白的转录和表达。结论 :成功构建了 pcDNA3.1( + ) -KGPcd真核表达载体并在哺乳动物细胞中能够正确转录和表达 ,为下一步作为基因疫苗免疫动物提供了依据。

AIM:To construct eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and to investigate the transcription and expression of recombined plasmid pcDNA3.1(+)/ KGPcd .METHODS:The pcDNA3.1(+)/ KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000.The transcription of pcDNA3.1(+)/ KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of pcDNA3.1(+)/ KGPcd was detected by method of indirect immunofluorescent.RESULTS:The eukaryotic expression vector of KGPcd gene was constructed successfully. It proved that the product of pcDNA3.1(+)/ KGPcd was expressed in transcripted COS7 cells.CONCLUSION:The recombinant plasmid pcDNA3.1(+)/ KGPcd could be transcripted and expressed correctly in mammalian cell COS7 in vitro .