利用琼脂磁珠快速筛选HIV-P24抗原核酸适配体

发布时间：2018-03-19 02:32

本文选题：琼脂磁珠　切入点：消减SELEX　出处：《西北师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：人类免疫缺陷病毒是艾滋病的病因。到目前为止,还没有安全有效的治疗办法,以预防为主,普及HIV抗原抗体及病毒的检测,尽可能早的发现感染者,早期干预,防止传播。其中,HIV-P24抗原是HIV的核心蛋白,在病毒的包装及成熟过程中起非常重要的作用。人体初次感染HIV,最早在患者血清中出现HIV抗原,2~3周可进行P24抗原的检测。HIV-P24抗原是能从血清中最早检测出的免疫标志物,HIV-P24蛋白早期准确检测有利于早期诊断,可以缩短“窗口期”,同时P24检测可预测病程,是跟踪HIV进程上的有效手段。因此,建立一种快速准确检测P24抗原的方法是很有必要的。适配体是人工体外筛选得到的一段寡核苷酸片段,由于其特殊的二级结构及三级结构,能与各种物质结合,如:抗原、抗体、病毒、金属离子等。他们之间的结合类似于抗原抗体的结合,因此,核酸适配体有“化学抗体”之称,但它有一些优于抗体的特性:高的亲合力和强的特异性;易制备及化学修饰;靶标范围广泛;分子小及无免疫原性。因此成功筛选出HIV-P24抗原特异性的适配体,有望建立一种基于该适配体的快速、准确、高效检测HIV-P24抗原的方法,可缩短检测的窗口期,为艾滋病的早期快速准确诊断奠定基础。以羧基化琼脂磁珠为靶标结合介质,HIV-P24抗原为靶标分子,用消减SELEX技术筛选HIV-P24抗原的特异性寡核苷酸适配体;后期又将筛选富集的次级文库进行大肠杆菌DH5α转化,并挑取阳性克隆进行交错PCR检测及序列测定。在整个HIV-P24抗原特异性寡核苷酸适配体筛选过程中对实验中磁珠投入量、洗涤试剂及次数、结合时间、非靶标分子的替换等因素进行调整,最终确立了一套较为完整的高效筛选此类适配体的方案。利用消减SELEX技术,经过七轮的筛选,成功筛选获得了HIV-P24抗原的特异性寡核苷酸适配体。将筛选得到的适配体转化及克隆,挑取克隆团,通过交错PCR技术鉴定阳性克隆,并对其特异性进行鉴定,最终得到了5个与HIV-P24抗原有较强特异性的克隆,获得了测序结果。消减SELEX技术筛选到的HIV-P24抗原特异性寡核苷酸适配体,具有强的特异性,有望建立一种基于HIV-P24抗原特异性寡核苷酸适配体的快速、高效、准确检测HIV-P24抗原的方法,可缩短检测的窗口期,为艾滋病的早期快速准确诊断奠定基础。
[Abstract]:The human immunodeficiency virus (HIV) is the cause of AIDS. So far, there is no safe and effective treatment that focuses on prevention, popularizes the detection of HIV antigens, antibodies and viruses, detects people infected as early as possible, and early intervention. Prevent transmission. HIV P24 antigen is the core protein of HIV. In the packaging and maturation of the virus plays a very important role. Human first infection of HIV, the earliest HIV antigen can be detected in the patient's serum for 3 weeks to detect P24 antigen. HIV-P24 antigen can be the first detected from the serum of the immune label. Early and accurate detection of HIV P24 protein is helpful for early diagnosis. "window period" can be shortened, and P24 detection can predict the course of disease, which is an effective means to track the HIV process. It is necessary to establish a rapid and accurate method for the detection of P24 antigen. Aptamer is a fragment of oligonucleotide obtained by artificial screening in vitro. Because of its special secondary and tertiary structures, aptamer can bind to various substances, such as antigen. Antibodies, viruses, metal ions, etc. Their binding is similar to the binding of antigens and antibodies, so nucleic acid aptamers are known as "chemical antibodies", but it has some advantages over antibodies: high affinity and strong specificity; Easy preparation and chemical modification; wide range of targets; small molecules and no immunogenicity. Therefore, the successful screening of HIV-P24 antigen specific aptamer is expected to establish a rapid, accurate and efficient method for detection of HIV-P24 antigen based on the aptamer. It can shorten the window period of detection and lay a foundation for the early rapid and accurate diagnosis of AIDS. The specific oligonucleotide aptamer of HIV-P24 antigen was screened by subtractive SELEX with carboxylated Agar beads as target binding medium and HIV-1 P24 antigen as target molecule. In the later stage, the enriched secondary library was transformed into Escherichia coli DH5 伪, and the positive clones were selected for interleaving PCR detection and sequencing. The amount of magnetic beads was injected into the whole HIV-P24 antigen specific oligonucleotide aptamer screening process. The washing reagents and times, combined with time, substitution of non-target molecules and other factors were adjusted to establish a relatively complete and efficient program for screening these aptamers. By using subtractive SELEX technology, seven rounds of screening were carried out. The specific oligonucleotide aptamer of HIV-P24 antigen was successfully screened. The selected aptamer was transformed and cloned. The positive clones were identified by interleaving PCR technique and their specificity was identified. Finally, five clones with strong specificity to HIV-P24 antigen were obtained, and sequencing results were obtained. HIV-P24 antigen-specific oligonucleotide aptamers screened by subtractive SELEX technique had strong specificity. It is expected that a rapid, efficient and accurate method for detecting HIV-P24 antigen based on HIV-P24 antigen-specific oligonucleotide aptamers can shorten the window period of detection and lay a foundation for the early rapid and accurate diagnosis of AIDS.
【学位授予单位】：西北师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R512.91

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