本文選題：卡波氏肉瘤相關皰疹病毒 + 血清流行病學　； 參考：《中國科學院研究生院（武漢病毒研究所）》2007年碩士論文

【摘要】： 卡波氏肉瘤相關皰疹病毒(Kaposi's sarcoma associated herpesvirus，KSHV)是新近發(fā)現(xiàn)的人類皰疹病毒，又稱為人皰疹病毒8型(Human herpesvirus 8，HHV-8)。流行病學研究證實該病毒是卡波氏肉瘤(Kaposi's sarcoma，KS)、原發(fā)性滲出性淋巴瘤(Primary effusion lynphoma，PEL)及多中心卡氏病(Mμlticentre cattleman's disease，MCD)的病原體。由于卡波氏肉瘤(Kaposi's sarcoma，KS)是AIDS患者中最常見的腫瘤，也高發(fā)于器官移植術后，因而作為KS病原體的KSHV的研究日益受到重視。 我國KSHV感染情況的研究以新疆維吾爾自治區(qū)為主，其KSHV感染率及KS發(fā)病率均較高，而我國其他省份報道不多。利用Hirt DNA提取法，，從KSHV潛伏感染細胞系BCBL-1中提取病毒DNA作為模板，通過特異引物PCR獲得病毒基因orf65及orf73。分別構建至原核表達載體，利用大腸桿菌表達系統(tǒng)表達His融合的病毒蛋白ORF65及ORF73羧基端多肽。通過Ni親和層析及電洗脫方法，我們獲得純化的ORF65蛋白及ORF73羧基端多肽用于開展以純化ORF65為抗原的的酶聯(lián)免疫吸附(ELISA)、免疫印記(western-blot)實驗，以ORF73羧基端多肽為抗原的酶聯(lián)免疫吸附實驗(EHSA)，結合以細胞BCBL-1為抗原的間接免疫熒光(IFA)實驗，檢測自2007年1月至2007年5月采集于湖北省高中學生的396份血漿樣本，并進行統(tǒng)計學分析。實驗發(fā)現(xiàn)，KSHV抗體陽性率為4.8％。不同性別間抗體陽性率差異無顯著性。 為后續(xù)實驗需要，我們以原核表達并純化的ORF73羧基端免疫大白兔，制備了多克隆抗體。該抗體可以識別原核表達的ORF73羧基端多肽，BCBL-1細胞核提取物中的病毒全長ORF73／LANA蛋白。在以BCBL-1細胞為抗原的IFA中能使細胞核著染典型的點狀熒光。 類似于其他的皰疹病毒，KSHV的生活史包含了裂解期與潛伏期。KSHV的潛伏感染與細胞轉化、腫瘤形成密切相關。目前用于治療KSHV感染的藥物主要針對病毒DNA多聚酶，能有效抑制病毒裂解復制，但無法清除潛伏病毒。本文探討了病毒蛋白潛伏期相關核抗原(Latency associated nuclear antigen，LANA)為藥物篩選靶標的可行性。我們合成了潛伏病毒中籍以結合LANA的LANA結合位點(LBS)DNA，經退火得到雙鏈LBS。將LBS插入載體序列中，利用同尾酶的特性，使插入載體的LBS數(shù)目不斷累積，直至插入64個串聯(lián)排列的LBS序列。經電泳遷移率變換實驗(EMSA)驗證，原核表達系統(tǒng)表達并純化的LANA羧基端多肽可以同合成的LBS結合，這種結合是特異性的。將含有串聯(lián)多聚LANA結合位點DNA(LBS)的質粒轉染潛伏感染KSHV的細胞系BCBL-1，連續(xù)觀察取樣，以Hirt技術提取樣品中的病毒DNA，通過熒光定量PCR(quantative PCR，qPCR)及常規(guī)PCR技術檢測細胞中病毒附加體數(shù)目的變化。實驗發(fā)現(xiàn)，串聯(lián)重復LBS通過競爭結合LANA，抑制LANA蛋白與病毒DNA的結合，阻斷LABIA功能的正常發(fā)揮，能夠減少潛伏于宿主細胞的病毒附加體。這為以LANA為靶點的藥物篩選提供了理論基礎。在此基礎上，我們構建了以病毒潛伏期相關核抗原(LANA)為靶標的高通量藥物篩選模型。將原核表達并經純化的LANA羧基端多肽吸附于96孔酶標板，通過比較加入藥物與未加藥物時，生物素標記的LBS與LANA的結合情況來判定藥物的有效性。生物素標記的LBS與LANA的結合情況可以通過加入辣根過氧化物酶(HRP)標記的鏈酶親和素及底物鄰苯二氨(OPD)-過氧化氫(H_2O_2)產生的顯色反應強度予以檢測。
[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).
【學位授予單位】：中國科學院研究生院（武漢病毒研究所）
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373;R965

【共引文獻】

相關期刊論文 前10條

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相關博士學位論文 前3條

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相關碩士學位論文 前7條

1 徐宏慧;蕈樣肉芽腫與HSV-1、HSV-2、EBV、HCMV及HHV-8相關性的研究[D];中國醫(yī)科大學;2002年

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6 曾妍;14-3-3β基因與卡波氏肉瘤相關性的研究[D];石河子大學;2007年

7 張德志;人類皰疹病毒8型的K1和K15亞型與新疆Kaposi肉瘤的相關性研究[D];石河子大學;2007年

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