本文選題：卡波氏肉瘤相關(guān)皰疹病毒 + 血清流行病學(xué)��； 參考：《中國(guó)科學(xué)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(guān)皰疹病毒(Kaposi's sarcoma associated herpesvirus，KSHV)是新近發(fā)現(xiàn)的人類(lèi)皰疹病毒，又稱(chēng)為人皰疹病毒8型(Human herpesvirus 8，HHV-8)。流行病學(xué)研究證實(shí)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(jiàn)的腫瘤，也高發(fā)于器官移植術(shù)后，因而作為KS病原體的KSHV的研究日益受到重視。 我國(guó)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(guó)其他省份報(bào)道不多。利用Hirt DNA提取法，，從KSHV潛伏感染細(xì)胞系BCBL-1中提取病毒DNA作為模板，通過(guò)特異引物PCR獲得病毒基因orf65及orf73。分別構(gòu)建至原核表達(dá)載體，利用大腸桿菌表達(dá)系統(tǒng)表達(dá)His融合的病毒蛋白ORF65及ORF73羧基端多肽。通過(guò)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(kāi)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(shí)驗(yàn)，以O(shè)RF73羧基端多肽為抗原的酶聯(lián)免疫吸附實(shí)驗(yàn)(EHSA)，結(jié)合以細(xì)胞BCBL-1為抗原的間接免疫熒光(IFA)實(shí)驗(yàn)，檢測(cè)自2007年1月至2007年5月采集于湖北省高中學(xué)生的396份血漿樣本，并進(jìn)行統(tǒng)計(jì)學(xué)分析。實(shí)驗(yàn)發(fā)現(xiàn)，KSHV抗體陽(yáng)性率為4.8％。不同性別間抗體陽(yáng)性率差異無(wú)顯著性。 為后續(xù)實(shí)驗(yàn)需要，我們以原核表達(dá)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(shí)別原核表達(dá)的ORF73羧基端多肽，BCBL-1細(xì)胞核提取物中的病毒全長(zhǎng)ORF73／LANA蛋白。在以BCBL-1細(xì)胞為抗原的IFA中能使細(xì)胞核著染典型的點(diǎn)狀熒光。 類(lèi)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(xì)胞轉(zhuǎn)化、腫瘤形成密切相關(guān)。目前用于治療KSHV感染的藥物主要針對(duì)病毒DNA多聚酶，能有效抑制病毒裂解復(fù)制，但無(wú)法清除潛伏病毒。本文探討了病毒蛋白潛伏期相關(guān)核抗原(Latency associated nuclear antigen，LANA)為藥物篩選靶標(biāo)的可行性。我們合成了潛伏病毒中籍以結(jié)合LANA的LANA結(jié)合位點(diǎn)(LBS)DNA，經(jīng)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(gè)串聯(lián)排列的LBS序列。經(jīng)電泳遷移率變換實(shí)驗(yàn)(EMSA)驗(yàn)證，原核表達(dá)系統(tǒng)表達(dá)并純化的LANA羧基端多肽可以同合成的LBS結(jié)合，這種結(jié)合是特異性的。將含有串聯(lián)多聚LANA結(jié)合位點(diǎn)DNA(LBS)的質(zhì)粒轉(zhuǎn)染潛伏感染KSHV的細(xì)胞系BCBL-1，連續(xù)觀察取樣，以Hirt技術(shù)提取樣品中的病毒DNA，通過(guò)熒光定量PCR(quantative PCR，qPCR)及常規(guī)PCR技術(shù)檢測(cè)細(xì)胞中病毒附加體數(shù)目的變化。實(shí)驗(yàn)發(fā)現(xiàn)，串聯(lián)重復(fù)LBS通過(guò)競(jìng)爭(zhēng)結(jié)合LANA，抑制LANA蛋白與病毒DNA的結(jié)合，阻斷LABIA功能的正常發(fā)揮，能夠減少潛伏于宿主細(xì)胞的病毒附加體。這為以LANA為靶點(diǎn)的藥物篩選提供了理論基礎(chǔ)。在此基礎(chǔ)上，我們構(gòu)建了以病毒潛伏期相關(guān)核抗原(LANA)為靶標(biāo)的高通量藥物篩選模型。將原核表達(dá)并經(jīng)純化的LANA羧基端多肽吸附于96孔酶標(biāo)板，通過(guò)比較加入藥物與未加藥物時(shí)，生物素標(biāo)記的LBS與LANA的結(jié)合情況來(lái)判定藥物的有效性。生物素標(biāo)記的LBS與LANA的結(jié)合情況可以通過(guò)加入辣根過(guò)氧化物酶(HRP)標(biāo)記的鏈酶親和素及底物鄰苯二氨(OPD)-過(guò)氧化氫(H_2O_2)產(chǎn)生的顯色反應(yīng)強(qiáng)度予以檢測(cè)。
[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(xué)位授予單位】：中國(guó)科學(xué)院研究生院（武漢病毒研究所）
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R373;R965

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張錫寶;卡波西肉瘤與HHV-8[J];嶺南皮膚性病科雜志;2001年02期

2 朱建中,盧春;人皰疹病毒8型K12基因在大腸桿菌中的克隆和表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2002年04期

3 曾怡,盧春,黃麗;人類(lèi)皰疹病毒8型包膜糖蛋白編碼基因K8.1的分離、克隆及在大腸桿菌中的表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年02期

4 黃麗,盧春,曾怡;卡波濟(jì)肉瘤相關(guān)皰疹病毒潛伏相關(guān)核抗原編碼基因的克隆及表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年03期

5 張心聲,齊眉,朱海峰,司桂玲,趙蔚明;230例健康獻(xiàn)血者卡波氏肉瘤相關(guān)皰疹病毒感染的血清學(xué)檢測(cè)[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2004年06期

6 李冬妹,楊磊,仙玲玲,譚曉華,李鋒,應(yīng)康,普雄民,王小波,何玲;用抑制性差減雜交構(gòu)建新疆Kaposi肉瘤差異表達(dá)基因文庫(kù)[J];中國(guó)生物工程雜志;2005年08期

7 付涌水;鄭優(yōu)榮;管洪宇;汪傳喜;江朝富;;廣州地區(qū)無(wú)償獻(xiàn)血人群人類(lèi)皰疹病毒8型血清流行病學(xué)調(diào)查[J];現(xiàn)代預(yù)防醫(yī)學(xué);2006年02期

8 程恩成;李漢忠;;移植相關(guān)性Kaposi肉瘤的研究進(jìn)展[J];國(guó)際移植與血液凈化雜志;2006年02期

9 譚曉華,楊磊;病毒感染在卡波氏肉瘤發(fā)病中的作用研究進(jìn)展[J];中國(guó)公共衛(wèi)生;2004年12期

10 譚曉華;李冬妹;狄春紅;楊磊;何玲;普雄明;;卡波氏肉瘤患者8型皰疹病毒套式PCR檢測(cè)[J];中國(guó)公共衛(wèi)生;2005年12期

相關(guān)博士學(xué)位論文 前3條

1 何方平;人類(lèi)8型皰疹病毒組合抗原血清檢測(cè)方法的建立及新疆地區(qū)流行病學(xué)調(diào)查[D];新疆醫(yī)科大學(xué);2006年

2 齊眉;人皰疹病毒8型重組蛋白的制備和應(yīng)用及其感染的流行病學(xué)調(diào)查[D];山東大學(xué);2006年

3 朱彪;我國(guó)人群KSHV感染狀態(tài)和分子流行病學(xué)研究及抗KSHV藥物的體外評(píng)價(jià)和篩選[D];浙江大學(xué);2008年

相關(guān)碩士學(xué)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(guān)性的研究[D];中國(guó)醫(yī)科大學(xué);2002年

2 黃麗;KSHV潛伏與增殖相關(guān)基因的克隆表達(dá)及其表達(dá)產(chǎn)物的免疫原性分析[D];南京醫(yī)科大學(xué);2004年

3 譚曉華;新疆卡波氏肉瘤血清流行病學(xué)研究[D];石河子大學(xué);2005年

4 李冬妹;用抑制性差減雜交篩選Kaposi肉瘤相關(guān)基因[D];石河子大學(xué);2005年

5 張心聲;人皰疹病毒8型（HHV-8）的基因表達(dá)及流行病學(xué)調(diào)查研究[D];山東大學(xué);2005年

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

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

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