Ag85A真核表达重组体的构建及稳定转染细胞系的建立

Construction of Eukaryotic Expression Recombinant Ag85A and Establishment of Stably Transfected Cell Line

构建结核杆菌抗原85A(Ag85A)的真核表达重组体,转染L929细胞,建立稳定转染细胞系。从质粒V1Jns.tPA-Ag85A中经PCR扩增出Ag85A基因,利用DNA重组技术将其插入到真核表达载体pcDNA3.1/myc-HisA中,经酶切和测序鉴定后,脂质体转染法转染L929细胞,通过G418选择培养,建立稳定转染细胞系,Western Blot检测Ag85A的表达。成功构建pcDNA3.1/myc-HisA-Ag85A真核表达载体并稳定转染L929细胞,成功表达了目的基因。为进一步研究Ag85ADNA疫苗对结核杆菌的免疫防护作用奠定了基础。

In order to construct a eukaryotic expression recombinant for Mycobacterium tuberculosis （Mtb） antigen 85A （Ag85A） and establish cell line L929 stably expressing it, gene AG85A was amplified by PCR from plasmid VIJns. tPA-Ag85A and cloned directly into plasmid pcDNA3, 1/myc-HisA. After identification by endonuclease diges- tion and DNA sequencing, the recombinant plasmid was transfected into L929 cell line by lipofectamine 2000. The stable transfectants were screened and cultured through G418 and were identified by Western blot. The results showed that the eukaryotic expression recombinant pcDNA3.1/myc-HisA-Ag85A was successfully constructed and the cell line stably expressed Ag85A was established. The establishment of cell line stably expressed Ag85A and the eukaryotic expression recombinant plasmid provide a solid experimental foundation for further research about the immunological protection of DNA vaccine against Mtb infection.