我国人群KSHV感染状态和分子流行病学研究及抗KSHV药物的体外评价和筛选

发布时间：2018-05-06 09:15

本文选题：卡波氏肉瘤相关疱疹病毒(KSHV) + 免疫缺陷　；参考：《浙江大学》2008年博士论文

【摘要】： 卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus KSHV),又名人类疱疹病毒-8(HHV-8),1994年由美国哥伦比亚大学病理学家Chang等用代表性差异分析法(representational different analysis)首先在卡波氏肉瘤组织中发现,国际上在KSHV病理学、流行病学以及相关分子生物学与致病机制研究方面已取得许多重要进展。研究发现KSHV感染与临床上卡波氏肉瘤(KS)、原发性渗出性淋巴瘤(PEL)、多中心Castleman's病(MCD)有关。 KSHV感染有一定的民族、地区差异,有关KSHV感染在中国境内的流行病学调查数据还十分有限,有关我国KSHV感染株的特点和致病性的研究报道更为少见,本研究对我国(浙江境内)不同人群感染状态和感染特点,特别是免疫缺陷者KSHV感染及流行株作一研究,并对抗潜伏感染态KSHV的药物进行了探索性研究。第一部分KSHV ORF65基因的克隆及重组蛋白的表达纯化根据Genbank GI:87196820序列,全基因合成258bp ORF65片段,对应氨基酸位于86aa—170aa,于EcoRV位点处插入PUC57,用EcoRⅠ和HindⅢ切下342bp片断,重组入PET44a原核表达质粒,转化DE3大肠杆菌,表达重组蛋白,纯化后获得0.7ug/ul纯度的ORF65重组蛋白,Western Blot显示其能识别KSHV感染病人血清。结论:我们获得了分子量大约75KD的KSHV ORF65重组蛋白,为我国开展KSHV感染的血清学研究提供了有效方法。第二部分KSHV ELISA法的建立和我国部分人群KSHV感染状态和感染特点研究 KSHV ELISA法的建立:用1μg/ml KSHV ORF65重组蛋白包被微孔反应板(每孔100ul),待检血浆及阳性、阴性对照血清用PBS-T 1:100稀释后每孔加100μl,辣根过氧化物酶标记的羊抗人1gG抗体为二抗,四甲基联苯胺(TMB)显色,450nm处测吸光度(A)值,以KSHV阴性对照血清的平均A450值加3倍标准差(STD)为临界值;在KSHV ORF26基因区段设计套式引物和Taq man探针,建立KSHV-DNA的套式检测和定量检测法,对我国部分经血传播高危人群,静脉吸毒者,肾移植受者,主要经血传播肝炎患者;经性传播高危人群、非静脉吸毒者、器官移植受者(肾移植受者,肝移植受者)、HIV感染者(抗病毒治疗者,未抗病毒治疗者)、普通人群进行KSHV感染状态和特点研究,并对慢性乙肝患者中KSHV感染状态与HBV复制、治疗因素进行相关性分析,研究结果数据见论文正文。研究结果提示,KSHV感染普遍存在于我国一般人群中;经血传播可能是我国KSHV感染的主要途径;艾滋病人和器官移植受者存在较高的KSHV感染,感染者有较高的活动性KSHV复制;抗HIV病毒治疗HAART可抑制KSH7复制;KSHV感染率在不同年龄段的慢乙肝患者感染率不同,以31～40岁年龄段为最高(37.1%):甘草类制剂在体内可能有抑制KSHV复制的作用。第三部分我国免疫缺陷者(浙江境内)KSHV流行株的进化树分析用基于KSHV ORF26基因的亚型分析方法,套式PCR获得172bp KSHV ORF26基因片段,纯化后用套式PCR的内套引物(P3,P4)双向测序,测序结果用Contig Express软件进行序列拼接获得完整序列,随机地对序列拼接后的13条序列采用Clustal 1.81程序软件进行基因进化分析,并与基因库中国际上流行的10株KSHV病毒株AY043000,AY042999,AY219458,AY219457,AY707887,AY707886,AY735096,AY735095,AY736237,AY736236进行基因同源性和进化树分析比较,用TreeView软件生成基因进化分析图,序列上传Genbank数据库,登录号为:EU026382;EU089809---EU089818,这是我国首次上传的有关KSHV亚型数据。研究结果表明,我国浙江境内的KSHV毒株的ORF26基因与匈牙利株AY707887最接近。属于A3亚型,A3亚型感染常不引起临床卡波氏肉瘤,研究结果提示了我国免疫缺陷人群卡波氏肉瘤发病率低的可能因素。第四部分抗KSHV药物的体外评价和筛选临床上抗疱疹病毒化学药物以核苷类似物为代表,它们通过抑制疱疹病毒的DNA多聚酶而抑制病毒复制,目前缺乏抗潜伏感染态KSHV的有效药物。第二部分的临床病例研究提示,甘草类制剂(美能或甘利欣)在体内可能有抗KSHV的作用。我们以BC-3细胞为模型,以KSHV-DNA实时荧光定量PCR测定为核心技术,用TPA(佛波酯)刺激BC-3细胞使KSHV进入溶细胞周期复制状态。以核苷类似物GCV(更昔洛韦)作对照,对GA(甘草酸)、ALLICIN(大蒜素)和EGCG(表没食子儿茶素没食子酸酯)进行了体外抗KSHV的研究,结果提示,GA、ALLICIN在体外对BC-3细胞内潜伏感染态KSHV环状DNA和KSHV病毒颗粒产生均由一定的抑制作用,GCV(更昔洛韦)和EGCG(表没食子儿茶素没食子酸酯)在体外对KSHV病毒颗粒产生有抑制作用,而对BC-3细胞内潜伏感染态KSHV环状DNA无明显的抑制作用。本部分研究结果表明,我们建立了以BC-3为细胞模型,以KSHV-DNA实时荧光定量PCR测定技术为核心的抗KSHV药物体外的评价体系,可成功用于抗KSHV的药物筛选;甘草酸和大蒜素在体外有抗潜伏感染态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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R512.99;R181.3;R965.1

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