卡波氏肉瘤相关人类疱疹病毒的流行病学调查及抗潜伏性感染药物筛选模型的建立

发布时间：2018-05-21 13:31

本文选题：卡波氏肉瘤相关疱疹病毒 + 血清流行病学　；参考：《中国科学院研究生院（武汉病毒研究所）》2007年硕士论文

【摘要】： 卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma associated herpesvirus，KSHV)是新近发现的人类疱疹病毒，又称为人疱疹病毒8型(Human herpesvirus 8，HHV-8)。流行病学研究证实该病毒是卡波氏肉瘤(Kaposi's sarcoma，KS)、原发性渗出性淋巴瘤(Primary effusion lynphoma，PEL)及多中心卡氏病(Mμlticentre cattleman's disease，MCD)的病原体。由于卡波氏肉瘤(Kaposi's sarcoma，KS)是AIDS患者中最常见的肿瘤，也高发于器官移植术后，因而作为KS病原体的KSHV的研究日益受到重视。我国KSHV感染情况的研究以新疆维吾尔自治区为主，其KSHV感染率及KS发病率均较高，而我国其他省份报道不多。利用Hirt DNA提取法，，从KSHV潜伏感染细胞系BCBL-1中提取病毒DNA作为模板，通过特异引物PCR获得病毒基因orf65及orf73。分别构建至原核表达载体，利用大肠杆菌表达系统表达His融合的病毒蛋白ORF65及ORF73羧基端多肽。通过Ni亲和层析及电洗脱方法，我们获得纯化的ORF65蛋白及ORF73羧基端多肽用于开展以纯化ORF65为抗原的的酶联免疫吸附(ELISA)、免疫印记(western-blot)实验，以ORF73羧基端多肽为抗原的酶联免疫吸附实验(EHSA)，结合以细胞BCBL-1为抗原的间接免疫荧光(IFA)实验，检测自2007年1月至2007年5月采集于湖北省高中学生的396份血浆样本，并进行统计学分析。实验发现，KSHV抗体阳性率为4.8％。不同性别间抗体阳性率差异无显著性。为后续实验需要，我们以原核表达并纯化的ORF73羧基端免疫大白兔，制备了多克隆抗体。该抗体可以识别原核表达的ORF73羧基端多肽，BCBL-1细胞核提取物中的病毒全长ORF73／LANA蛋白。在以BCBL-1细胞为抗原的IFA中能使细胞核着染典型的点状荧光。类似于其他的疱疹病毒，KSHV的生活史包含了裂解期与潜伏期。KSHV的潜伏感染与细胞转化、肿瘤形成密切相关。目前用于治疗KSHV感染的药物主要针对病毒DNA多聚酶，能有效抑制病毒裂解复制，但无法清除潜伏病毒。本文探讨了病毒蛋白潜伏期相关核抗原(Latency associated nuclear antigen，LANA)为药物筛选靶标的可行性。我们合成了潜伏病毒中籍以结合LANA的LANA结合位点(LBS)DNA，经退火得到双链LBS。将LBS插入载体序列中，利用同尾酶的特性，使插入载体的LBS数目不断累积，直至插入64个串联排列的LBS序列。经电泳迁移率变换实验(EMSA)验证，原核表达系统表达并纯化的LANA羧基端多肽可以同合成的LBS结合，这种结合是特异性的。将含有串联多聚LANA结合位点DNA(LBS)的质粒转染潜伏感染KSHV的细胞系BCBL-1，连续观察取样，以Hirt技术提取样品中的病毒DNA，通过荧光定量PCR(quantative PCR，qPCR)及常规PCR技术检测细胞中病毒附加体数目的变化。实验发现，串联重复LBS通过竞争结合LANA，抑制LANA蛋白与病毒DNA的结合，阻断LABIA功能的正常发挥，能够减少潜伏于宿主细胞的病毒附加体。这为以LANA为靶点的药物筛选提供了理论基础。在此基础上，我们构建了以病毒潜伏期相关核抗原(LANA)为靶标的高通量药物筛选模型。将原核表达并经纯化的LANA羧基端多肽吸附于96孔酶标板，通过比较加入药物与未加药物时，生物素标记的LBS与LANA的结合情况来判定药物的有效性。生物素标记的LBS与LANA的结合情况可以通过加入辣根过氧化物酶(HRP)标记的链酶亲和素及底物邻苯二氨(OPD)-过氧化氢(H_2O_2)产生的显色反应强度予以检测。
[Abstract]:The Kaposi's sarcoma associated herpesvirus (KSHV) is a newly discovered human herpesvirus, also known as the human herpesvirus 8 (Human herpesvirus 8, HHV-8). Epidemiological studies have confirmed that the virus is kapoy's sarcoma (Kaposi's sarcoma, KS), primary exudative lymphoma (Primary). PEL) and the pathogen of polycentric kapson disease (M lticentre cattleman's disease, MCD). As the most common tumor in AIDS patients, as Kaposi's sarcoma (KS) is the most common tumor in AIDS, it is also high in organ transplantation, so the study of KSHV as KS pathogen has been paid more and more attention.
The study of KSHV infection in China is mainly in the Xinjiang Uygur Autonomous Region, its KSHV infection rate and the incidence of KS are high, but there are not many reports in other provinces in China. Using the Hirt DNA extraction method, the virus DNA is extracted from the KSHV latent infection cell line BCBL-1 as a template, and the virus gene orf65 and orf73. are obtained by the specific primer PCR. The viral protein ORF65 and ORF73 carboxyl terminal polypeptide of His fusion were expressed by the expression system of Escherichia coli. By Ni affinity chromatography and electroelution, we obtained the purified ORF65 protein and the ORF73 carboxyl terminal polypeptide for the enzyme linked immunosorbent (ELISA), and the immune imprint (Western-blot) test for the purification of ORF65 as antigen. 396 plasma samples collected from high school students in Hubei province from January 2007 to May 2007 were detected by enzyme linked immunosorbent assay (EHSA) with ORF73 carboxyl terminus peptide as antigen and indirect immunofluorescence (IFA) experiment with cell BCBL-1 as antigen. The results showed that the positive rate of KSHV antibody was between 4.8%. and sex. There was no significant difference in the positive rate of antibody.
In order to follow up the experiment, we prepared the polyclonal antibody with the ORF73 carboxyl terminal of the prokaryotic expression and purification. The antibody can identify the ORF73 carboxyl terminal polypeptide expressed in the prokaryotic cell and the full length of the ORF73 / LANA protein in the BCBL-1 nuclear extract. The nucleus is infected with the typical point in the IFA of the BCBL-1 cell as the antigen of the antigen. Like fluorescence.
Similar to other herpes viruses, the life history of KSHV contains the latent infection of.KSHV in lysis and incubation period and cell transformation, and the formation of tumor is closely related. The current drug used for the treatment of KSHV infection is mainly directed against the virus DNA polymerase, which can effectively inhibit the replication of the virus, but the latent virus can not be scavenged. This paper discusses the latent virus protein potential. The Latency associated nuclear antigen (LANA) is the possibility of screening the target for the drug. We synthesized the LANA binding site (LBS) DNA in the latent virus, which combined LANA with the LANA binding site (LBS) DNA. 64 series of LBS sequences were introduced. The electrophoretic mobility transformation test (EMSA) showed that the LANA carboxyl terminal polypeptide expressed and purified by the prokaryotic expression system could be combined with the synthesized LBS. The binding was specific. The plasmid containing the DNA (LBS) containing the series of LANA binding site DNA (LBS) was transfected to the cell line of the latent infection KSHV. The virus DNA in the sample was extracted by Hirt, and the number of virus additional bodies in the cells was detected by fluorescence quantitative PCR (quantative PCR, qPCR) and routine PCR. The experiment found that the tandem repeat LBS could inhibit the normal play of LABIA function by combining competition with LANA, and blocking LANA protein and viral DNA, and could reduce the latency in lodging. This provides a theoretical basis for drug screening targeting LANA. On this basis, we constructed a high throughput drug screening model with LANA as a target. The prokaryotic and purified LANA carboxyl terminated polypeptide was attached to the 96 pore enzyme standard plate, and the drug was added by comparison. The binding of biotin labeled LBS and LANA was not added to determine the efficacy of the drug. The combination of biotin labeled LBS and LANA could be detected by the chromogenic reaction intensity produced by adding horseradish peroxidase (HRP) labeled streptavidin and substrate phthalate (OPD) - hydrogen peroxide (H_2O_2).
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373;R965

【共引文献】