摘要
目的:构建并鉴定表达HIV-1 CRF01_AE亚型结构基因的小鼠模型。方法:使用哺乳动物密码子优化的HIV-1 CRF01_AE gp160基因,通过慢病毒包装系统构建重组慢病毒LV-GFP-AE gp160,将上述重组慢病毒感染小鼠肺上皮细胞TC-1,经嘌呤霉素抗性筛选获得稳定表达gp160基因的TC-1细胞。采用RT-PCR、流式细胞术检测gp160基因在细胞内的表达稳定性,将稳定表达Gp160蛋白的TC-1-HIV AE gp160细胞接种小鼠,用免疫组化方法检测小鼠体内细胞团块中HIV Gp160蛋白的表达。结果:菌落PCR、酶切鉴定和测序表明重组质粒pLVX-AE gp160构建正确,RT-PCR、GFP荧光及流式细胞术结果均显示gp160基因能在细胞TC-1中稳定表达,免疫组化结果也表明小鼠体内接种的细胞可以稳定表达HIV Gp160蛋白。结论:建立了稳定表达HIV-1 CRF01_AE亚型Gp160蛋白的TC-1细胞及小鼠模型,为HIV-1 CRF01_AE亚型HIV疫苗的临床前研究提供了可靠的体外、体内免疫原性评价工具,为该疫苗的进一步开发奠定了坚实的实验基础。
Objective:To establish and identify a mouse model expressing the structural gene of HIV-1 CRF01_AE subtype.Methods:A recombinant lentivirus LV-GFP-AE gp160 was constructed by a lentiviral packaging system based on the mammalian codon optimized HIV-1 CRF01_AE gp160 gene, and was infected into mouse lung epithelial cells TC-1. The TC-1 cells stably expressing gp160 gene were obtained by anti-puromycin screening. RT-PCR and flow cytometry were used to detect the expression stability of gp160 gene in TC-1 cells.Then, TC-1-HIV AE gp160 cells were inoculated into mice, and the HIV Gp160 protein expression of cell mass in mice was detected by immunohistochemistry.Results:Colony PCR, restriction enzyme digestion and sequencing showed that the recombinant plasmid pLVX-AE gp160 was constructed correctly. RT-PCR, GFP fluorescence and flow cytometry results further confirmed that the gp160 gene could be stably expressed in TC-1 cells, and immunohistochemistry results showed the TC-1-HIV AE gp160 cells inoculated in mice can also stably express HIV Gp160 protein.Conclusion:Establishment of TC-1 cells and mouse model that stably expressed HIV-1 CRF01_AE subtype Gp160 protein provided reliable in vitro and in vivo immunogenicity evaluation tools for preclinical studies of HIV-1 CRF01_AE subtype HIV vaccine, and laid a solid experimental foundation for the further development of the vaccine.
作者
郭婷婷
岳成
徐柯
周玉柏
GUO Ting-Ting;YUE Cheng;XU Ke;ZHOU Yu-Bai(College of Life Science and Bioengineering,Beijing University of Technology,Beijing 10024;Institute for Vi-ral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China)
出处
《生物技术通讯》
CAS
2020年第3期257-261,共5页
Letters in Biotechnology
基金
艾滋病和病毒性肝炎等重大传染病防治国家科技重大专项(2018ZX10731101-002)。
