本文選題：卡波氏肉瘤相關(guān)皰疹病毒(KSHV) + 免疫缺陷　； 參考：《浙江大學(xué)》2008年博士論文

【摘要】： 卡波氏肉瘤相關(guān)皰疹病毒(Kaposi's sarcoma-associated herpesvirus KSHV),又名人類(lèi)皰疹病毒-8(HHV-8),1994年由美國(guó)哥倫比亞大學(xué)病理學(xué)家Chang等用代表性差異分析法(representational different analysis)首先在卡波氏肉瘤組織中發(fā)現(xiàn),國(guó)際上在KSHV病理學(xué)、流行病學(xué)以及相關(guān)分子生物學(xué)與致病機(jī)制研究方面已取得許多重要進(jìn)展。研究發(fā)現(xiàn)KSHV感染與臨床上卡波氏肉瘤(KS)、原發(fā)性滲出性淋巴瘤(PEL)、多中心Castleman's病(MCD)有關(guān)。 KSHV感染有一定的民族、地區(qū)差異,有關(guān)KSHV感染在中國(guó)境內(nèi)的流行病學(xué)調(diào)查數(shù)據(jù)還十分有限,有關(guān)我國(guó)KSHV感染株的特點(diǎn)和致病性的研究報(bào)道更為少見(jiàn),本研究對(duì)我國(guó)(浙江境內(nèi))不同人群感染狀態(tài)和感染特點(diǎn),特別是免疫缺陷者KSHV感染及流行株作一研究,并對(duì)抗?jié)摲腥緫B(tài)KSHV的藥物進(jìn)行了探索性研究。 第一部分KSHV ORF65基因的克隆及重組蛋白的表達(dá)純化 根據(jù)Genbank GI:87196820序列,全基因合成258bp ORF65片段,對(duì)應(yīng)氨基酸位于86aa—170aa,于EcoRV位點(diǎn)處插入PUC57,用EcoRⅠ和HindⅢ切下342bp片斷,重組入PET44a原核表達(dá)質(zhì)粒,轉(zhuǎn)化DE3大腸桿菌,表達(dá)重組蛋白,純化后獲得0.7ug/ul純度的ORF65重組蛋白,Western Blot顯示其能識(shí)別KSHV感染病人血清。結(jié)論:我們獲得了分子量大約75KD的KSHV ORF65重組蛋白,為我國(guó)開(kāi)展KSHV感染的血清學(xué)研究提供了有效方法。 第二部分KSHV ELISA法的建立和我國(guó)部分人群KSHV感染狀態(tài)和感染特點(diǎn)研究 KSHV ELISA法的建立:用1μg/ml KSHV ORF65重組蛋白包被微孔反應(yīng)板(每孔100ul),待檢血漿及陽(yáng)性、陰性對(duì)照血清用PBS-T 1:100稀釋后每孔加100μl,辣根過(guò)氧化物酶標(biāo)記的羊抗人1gG抗體為二抗,四甲基聯(lián)苯胺(TMB)顯色,450nm處測(cè)吸光度(A)值,以KSHV陰性對(duì)照血清的平均A450值加3倍標(biāo)準(zhǔn)差(STD)為臨界值;在KSHV ORF26基因區(qū)段設(shè)計(jì)套式引物和Taq man探針,建立KSHV-DNA的套式檢測(cè)和定量檢測(cè)法,對(duì)我國(guó)部分經(jīng)血傳播高危人群,靜脈吸毒者,腎移植受者,主要經(jīng)血傳播肝炎患者;經(jīng)性傳播高危人群、非靜脈吸毒者、器官移植受者(腎移植受者,肝移植受者)、HIV感染者(抗病毒治療者,未抗病毒治療者)、普通人群進(jìn)行KSHV感染狀態(tài)和特點(diǎn)研究,并對(duì)慢性乙肝患者中KSHV感染狀態(tài)與HBV復(fù)制、治療因素進(jìn)行相關(guān)性分析,研究結(jié)果數(shù)據(jù)見(jiàn)論文正文。 研究結(jié)果提示,KSHV感染普遍存在于我國(guó)一般人群中;經(jīng)血傳播可能是我國(guó)KSHV感染的主要途徑;艾滋病人和器官移植受者存在較高的KSHV感染,感染者有較高的活動(dòng)性KSHV復(fù)制;抗HIV病毒治療HAART可抑制KSH7復(fù)制;KSHV感染率在不同年齡段的慢乙肝患者感染率不同,以31～40歲年齡段為最高(37.1%):甘草類(lèi)制劑在體內(nèi)可能有抑制KSHV復(fù)制的作用。 第三部分我國(guó)免疫缺陷者(浙江境內(nèi))KSHV流行株的進(jìn)化樹(shù)分析 用基于KSHV ORF26基因的亞型分析方法,套式PCR獲得172bp KSHV ORF26基因片段,純化后用套式PCR的內(nèi)套引物(P3,P4)雙向測(cè)序,測(cè)序結(jié)果用Contig Express軟件進(jìn)行序列拼接獲得完整序列,隨機(jī)地對(duì)序列拼接后的13條序列采用Clustal 1.81程序軟件進(jìn)行基因進(jìn)化分析,并與基因庫(kù)中國(guó)際上流行的10株KSHV病毒株AY043000,AY042999,AY219458,AY219457,AY707887,AY707886,AY735096,AY735095,AY736237,AY736236進(jìn)行基因同源性和進(jìn)化樹(shù)分析比較,用TreeView軟件生成基因進(jìn)化分析圖,序列上傳Genbank數(shù)據(jù)庫(kù),登錄號(hào)為:EU026382;EU089809---EU089818,這是我國(guó)首次上傳的有關(guān)KSHV亞型數(shù)據(jù)。 研究結(jié)果表明,我國(guó)浙江境內(nèi)的KSHV毒株的ORF26基因與匈牙利株AY707887最接近。屬于A3亞型,A3亞型感染常不引起臨床卡波氏肉瘤,研究結(jié)果提示了我國(guó)免疫缺陷人群卡波氏肉瘤發(fā)病率低的可能因素。 第四部分抗KSHV藥物的體外評(píng)價(jià)和篩選 臨床上抗皰疹病毒化學(xué)藥物以核苷類(lèi)似物為代表,它們通過(guò)抑制皰疹病毒的DNA多聚酶而抑制病毒復(fù)制,目前缺乏抗?jié)摲腥緫B(tài)KSHV的有效藥物。第二部分的臨床病例研究提示,甘草類(lèi)制劑(美能或甘利欣)在體內(nèi)可能有抗KSHV的作用。我們以BC-3細(xì)胞為模型,以KSHV-DNA實(shí)時(shí)熒光定量PCR測(cè)定為核心技術(shù),用TPA(佛波酯)刺激BC-3細(xì)胞使KSHV進(jìn)入溶細(xì)胞周期復(fù)制狀態(tài)。以核苷類(lèi)似物GCV(更昔洛韋)作對(duì)照,對(duì)GA(甘草酸)、ALLICIN(大蒜素)和EGCG(表沒(méi)食子兒茶素沒(méi)食子酸酯)進(jìn)行了體外抗KSHV的研究,結(jié)果提示,GA、ALLICIN在體外對(duì)BC-3細(xì)胞內(nèi)潛伏感染態(tài)KSHV環(huán)狀DNA和KSHV病毒顆粒產(chǎn)生均由一定的抑制作用,GCV(更昔洛韋)和EGCG(表沒(méi)食子兒茶素沒(méi)食子酸酯)在體外對(duì)KSHV病毒顆粒產(chǎn)生有抑制作用,而對(duì)BC-3細(xì)胞內(nèi)潛伏感染態(tài)KSHV環(huán)狀DNA無(wú)明顯的抑制作用。本部分研究結(jié)果表明,我們建立了以BC-3為細(xì)胞模型,以KSHV-DNA實(shí)時(shí)熒光定量PCR測(cè)定技術(shù)為核心的抗KSHV藥物體外的評(píng)價(jià)體系,可成功用于抗KSHV的藥物篩選;甘草酸和大蒜素在體外有抗?jié)摲腥緫B(tài)KSHV作用。
[Abstract]:Kapo's sarcoma related herpes virus (Kaposi's sarcoma-associated herpesvirus KSHV), also known as human herpesvirus -8 (HHV-8), was first found in kapoe's sarcoma tissue by the representative differential analysis (representational different analysis) by pathologist Chang of the Columbia University in 1994. Many important advances have been made in epidemiology, related molecular biology and pathogenesis research. The study found that KSHV infection is associated with clinical Kaposi's sarcoma (KS), primary exudative lymphoma (PEL), and polycentric Castleman's disease (MCD).
KSHV infection has certain ethnic and regional differences, and the epidemiological survey data about KSHV infection in China are still very limited. The research reports on the characteristics and pathogenicity of the KSHV infected plants in China are more rare. This study has the characteristics of the infection state and infection of different populations in China (Zhejiang), especially the KSHV infection of the immunodeficiency patients and The epidemic strains were studied and the drugs against latent KSHV were explored.
Part 1 cloning of KSHV ORF65 gene and expression and purification of its recombinant protein
According to the Genbank GI:87196820 sequence, the whole gene was synthesized by the 258bp ORF65 fragment, the amino acid was located at 86aa - 170aa, and PUC57 was inserted at the EcoRV site. The recombinant plasmid was reorganized into the PET44a prokaryotic expression plasmid with EcoR I and Hind III, and the recombinant protein was expressed and purified. Blot shows that it can identify the serum of patients with KSHV infection. Conclusion: we have obtained a recombinant protein of KSHV ORF65 with a molecular weight of about 75KD, which provides an effective method for the serological study of KSHV infection in China.
The second part is the establishment of KSHV ELISA and the characteristics of KSHV infection and infection in some Chinese population.
The establishment of KSHV ELISA method: the recombinant protein of 1 mu g/ml KSHV ORF65 was wrapped by a microporous reaction plate (100ul per pore), and the plasma and positive were detected. The negative control serum was diluted by PBS-T 1:100 with 100 micron. The anti human 1gG antibody labeled by horseradish peroxidase was two, four methylaniline (TMB) was coloured, and the value of absorbance was negative. The average A450 value of the serum and the 3 times standard deviation (STD) was the critical value. A set primer and a Taq man probe were designed at the KSHV ORF26 gene section to establish a nested detection and quantitative detection method for KSHV-DNA, which was used in some high risk groups, intravenous drug users, renal transplant recipients, major blood transmitted hepatitis patients, and high risk sexually transmitted people. Non intravenous drug users, organ transplant recipients (renal transplant recipients, liver transplant recipients), HIV infected persons (antiviral treatment, non antiviral), general population KSHV infection status and characteristics of the study, and chronic hepatitis B patients with KSHV infection status and HBV replication, the correlation analysis of the treatment factors, the results of the research results of the paper text.
The results suggest that KSHV infection is common in the general population of our country; the blood transmission may be the main way of KSHV infection in China; the patients with AIDS and organ transplant recipients have higher KSHV infection, the infected persons have high active KSHV replication, and the anti HIV virus treatment HAART can inhibit the KSH7 replication; the infection rate of KSHV is slow in different age groups. The infection rate of hepatitis B patients is different. The highest age is 31~40 years old (37.1%): licorice preparations may inhibit KSHV replication in vivo.
The third part is the evolutionary tree analysis of KSHV epidemic strains in Chinese immunodeficiency patients (Zhejiang).
Using the subtype analysis method based on the KSHV ORF26 gene, the 172bp KSHV ORF26 gene fragment was obtained by the set of PCR. After purification, the nested primers (P3, P4) of the nested PCR were sequenced, and the sequence was sequenced with Contig Express software, and the 13 sequences after the sequence were randomly selected as the Clustal 1.81 program software. Based on evolutionary analysis, the genetic homology and evolutionary tree analysis of 10 KSHV virus strains AY043000, AY042999, AY219458, AY219457, AY707887, AY707886, AY735096, AY735095, AY736237, and AY736236 were compared with the internationally popular strains of the gene bank. 2, EU089809---EU089818, this is the first upload of KSHV subtype data in China.
The results show that the ORF26 gene of the KSHV strain in Zhejiang is the closest to the Hungarian strain AY707887. It belongs to the A3 subtype, and the A3 subtype infection often does not cause clinical Kapo sarcoma. The results suggest the possible factors for the low incidence of Kaposi's sarcoma in the immune deficient population of our country.
In vitro evaluation and screening of fourth parts of anti KSHV drugs
The clinical antiherpesvirus chemicals are represented by nucleoside analogues. They inhibit the replication of the virus by inhibiting the DNA polymerase of herpes virus and currently lack the effective drugs to resist the latent infection state KSHV. The second part of clinical case study suggests that Glycyrrhiza (energy or Gan Lixin) may have the effect of anti KSHV in the body. Taking BC-3 cells as the model, using KSHV-DNA real-time fluorescence quantitative PCR determination as the core technology, BC-3 cells were stimulated by TPA (phorbol ester) to enter the cell cycle replicative state. The anti GA (glycyrrhizic acid), ALLICIN (allicin) and EGCG (epigallocatechin gallate) were carried out in vitro anti KSHV in vitro, with BC-3 cells stimulated by TPA (phorbol ester). The results suggest that GA, ALLICIN can inhibit the latent infection of KSHV cyclic DNA and KSHV virus particles in BC-3 cells in vitro, and GCV (ganciclovir) and EGCG (epigallocatechin gallate) have inhibitory effect on KSHV virus particles in vitro, and KSHV annular DNA in BC-3 cells The results show that we have established an in vitro evaluation system of anti KSHV drugs with BC-3 as the cell model and KSHV-DNA real-time fluorescence quantitative PCR determination technology as the core. It can be successfully used in the screening of anti KSHV drugs, and the effect of glycyrrhizic acid and Allicin against the latent infection state of KSHV in vitro.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R512.99;R181.3;R965.1

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相關(guān)期刊論文 前1條

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