HBV共价闭合环状DNA功能化纳米颗粒制备及鉴定

摘要

目的 制备HBV共价闭合环状DNA(cccDNA)功能化磁性纳米颗粒,为进一步建立富集分离和检测cccDNA方法奠定基础. 方法 通过亲和素修饰纳米颗粒表面,利用亲和素与生物素的亲和作用,将亲和素修饰的纳米颗粒与生物素标记的cccDNA特异性探针偶联,制备cccDNA功能化纳米颗粒,最后用cccDNA探针完全互补的荧光标记序列与功能化纳米颗粒反应,鉴定其特性. 结果 亲和素修饰的纳米颗粒能结合生物素最大量为1 205 ng/mg;通过与亲和素作用,生物素标记cccDNA特异性探针固定在磁性纳米微粒表面,其最大结合生物素标记探针量为3.2×10-9 mol/mg;cccDNA功能化纳米颗粒可捕获与其互补的荧光标记寡核苷酸序列,其最大捕获量为2.0×10-9mol/mg,并能通过变性释放出保持生物学活性游离的荧光标记寡核苷酸序列. 结论 成功构建的cccDNA功能化纳米微粒能有效捕获与其互补的序列,其捕获量能达到cccDNA的分离提取要求.%Objective To prepare and identify specific nanoparticles of HBV cccDNA,and lay the foundation for enriching,separating,and detecting cccDNA in the future.Methods Initially,the silicacoated nanoparticles were modified by streptavidin.Then oligonucleotides for specific HBV cccDNA capture were designed and labeled by biotin.Moreover,oligonucleotides labeled with fluorescein isothiocyanate (FITC),which were completely complementary to capture probe,were synthesized and captured by functionalized nanoparticles for the characteristic validation.Results Streptavidin-modified nanoparticles could bind biotin,its maximum amount was about 1 215 ng/mg.In addition,streptavidin-modified nanoparticles had a high oligonucleotides binding capacity,its average amount of nucleic acid fixed was about 3 200 pmol/mg.After FITC-labeled sequences were added to the buffer and its hybridization with functionalized nanoparticles was performed,the fluorescence intensity of supernatant was decreased noticeably compared with that of pre-hybridization.Moreover,functionalized nanoparticles could capture up to about 2 000 pmol FITC-labeled sequences.Conclusion Specific functionalized nanoparticles of cccDNAcan effectively capture the complementary sequences labeled by FITC,and the capture amount is enough to capture cccDNA in routine samples.