昆明地区HCV分子流行病学研究及Core蛋白表达质粒构建

发布时间：2018-02-16 07:15

本文关键词： 丙型肝炎病毒基因型基因亚型 5’NCR-C区核心蛋白诊断检测　出处：《昆明理工大学》2007年硕士论文　论文类型：学位论文

【摘要】： 丙型肝炎病毒(hepatitis C virus，HCV)呈全球分布，自然感染率约为3％，即有1.7～2.0亿人为丙型肝炎感染者。近年来，HCV在部分地区的流行呈增长态势，我国的HCV估计感染率为2.5-4.9％，感染总人数超过5000万人。HCV一经感染，大部分感染者将发展成慢性丙型肝炎，并有较大比例的感染者最终发展成肝硬化、肝癌。由于缺乏可以治愈的药物和疫苗，对丙型肝炎尚不能有效的治疗与预防，丙型肝炎已经成为继艾滋病之后的又一大严重危害人类健康的传染病。由于HCV的依赖RNA的RNA聚合酶(RdRp)缺乏校对功能，导致HCV基因突变频率极高。根据HCV基因组核苷酸序列的同源性，HCV可被分为6个基因型，70多个亚型，以及众多的准种。HCV基因型分布呈现明显的区域差异；丙型肝炎患者的临床症状、病程进展，及药物治疗效果均与基因型密切相关。HCV基因型的研究有助于明确HCV流行的危险因素与传播途径，了解病毒的变异、进化特点，并为地方性疫苗的研制，药物的研究与开发，患者临床观察与治疗措施的制定提供流行病学依据。对HCV感染的早期诊断是控制HCV传播的重要手段，其中HCV核心抗原的检测对处于HCV感染“窗口期”的个体检测有很大价值。本研究经对采自云南省昆明市42例非吸毒所致丙型肝炎患者样品，和14例静脉吸毒所致HCV／HIV共感染者的样品，进行了血浆病毒核酸提取，HCV 5’NCR-C区基因的核苷酸序列测定与分析。发现： 1．本研究建立的基于5’NCR-C区的基因分型方法可以将所有待测样本有效分型，弥补了5’NCR区在基因分型方法不能区分1型和6型固有缺陷。 2．昆明市非静脉吸毒所致丙型肝炎病人中，1b亚型仍为主要流行型，其次为3型，2a亚型也有一定比例的流行，同时发现有6k和6n亚型，这与HCV在我国其它省份普通人群中1b／2a为主的基因型分布明显不同。 3．昆明市静脉吸毒人群中HCV感染者中以1b和3为主，同时发现有6n，没有发现2a亚型，此种HCV基因型分布特点与东南亚国家更为相似。 4．本研究中普通人群1b型的组成更为复杂，可被分为五个组，6例静脉吸毒1b型样品则全部归入第二个组中，证明昆明市普通人群HCV传入呈多途径方式，但对静脉吸毒人群则以单途径方式传入。 5．HCV C区HLAA2型CTL抗原表位在1b与3b型之间有明显差异，可能是1b亚型易于慢性感染并快速致病原因之一。 6．丙型肝炎病程与ALT、AST等肝功生化指标之间无显著相关性，不同基因型间ALT、AST水平无明显差异，但1b型患者HCV病毒载量显著高于其它基因型患者，与AST，ALT等肝功生化指标相比较，HCV病毒载量更适合作为丙型肝炎病程评价指标。对丙型肝炎早期诊断是控制HCV传播的重要手段，其中基于HCV核心蛋白及相关抗体反应的检测方法对HCV感染者“窗口期”诊断有很大价值，Core蛋白表达是建立此方法的核心步骤。本研究将HCV Core基因扩增产物克隆到pET 100／D-TopoVector，在BL21Chemically Competent Ecoli中表达，进而用SDS-PAGE电泳检测，发现：经克隆获得了正确的Core基因，并可诱导表达出预期大小的蛋白分子(约23KD)，在诱导培养2.5小时，可获得最大表达量。综上所述，本研究对云南省昆明市丙型肝炎患者的HCV基因型分布进行了较为系统的研究，所取得结果将有助于明确云南省HCV分子流行病学特点、追溯HCV病毒传播来源，分析HCV病毒进化特点，同时为我省丙型肝炎患者病人临床诊断与治疗，及进行药物研发合地方性疫苗研制提供前期研究基础。HCV Core蛋白表达构建将为研制高特异性和敏感性的HCV抗体诊断试剂奠定基础。
[Abstract]:Hepatitis C virus (hepatitis C, virus, HCV) is a global distribution, natural infection rate is about 3%, which is 1.7 ~ 200 million for HCV infection. In recent years, HCV growth in some areas of the epidemic, China's HCV estimated the infection rate was 2.5-4.9%, the total number of infections over 50 million people.HCV after infection, most infected people will develop chronic hepatitis C infection, and a larger proportion of eventually develop cirrhosis, liver cancer can be cured. Due to the lack of drugs and vaccines for hepatitis C can effectively treat and prevent hepatitis C, AIDS has become the following after another serious infection disease.
Due to the reliance on RNA HCV RNA (RdRp) polymerase lacks proofreading function, leading to HCV gene mutation frequency is very high. According to the homology of HCV nucleotide sequence, HCV can be divided into 6 genotypes, more than 70 genotypes,.HCV genotype and quasispecies distribution showed numerous regional differences; clinical progress the symptoms of hepatitis C patients, risk factors and the effect of chemotherapy are closely related with the genotype.HCV genotype will help identify the prevalence of HCV and dissemination of knowledge of virus mutation, evolution characteristics, and for the development of local research and development of vaccines, drugs, clinical observation and treatment of patients provide basis for the formulation of epidemiology. The early diagnosis of HCV infection is an important means to control the spread of HCV, the detection of HCV core antigen in HCV infected individual detection window period has great value.
This study was carried out on 42 samples of hepatitis C patients caused by non drug use and 14 cases of HCV / HIV co infection caused by intravenous drug users in Kunming city of Yunnan province. The nucleotide sequence of HCV 5 'NCR-C gene was detected and analyzed.
1., based on the 5 'NCR-C region, the genotyping method established in this study can effectively classify all the tested samples, and make up for the 5' NCR area. The genotyping method can't distinguish between 1 and 6 types of inherent defects.
The city of Kunming in 2. non hepatitis C patients caused by intravenous drug users, 1b subtype is the major epidemic type, followed by type 3, 2A subtypes also have a certain proportion of popular, also found 6K and 6N subtypes, and the HCV in the general population of other provinces in China in the 1B / 2A genotype distribution obviously different.
3. among the HCV patients in Kunming, 1b and 3 were the main causes of 6N infection. Meanwhile, 6N and 2A subtypes were not found. The distribution characteristics of this HCV genotype were more similar to those in Southeast Asian countries.
4., in the study, the composition of 1B in the general population is more complex, and can be divided into five groups. 6 cases of intravenous drug abuse 1b type are all classified into second groups. It is proved that the HCV afferents of the general population in Kunming have multiple ways, but the intravenous drug users are introduced in a single way.
The HLAA2 type CTL antigen epitopes of 5.HCV C region have obvious differences between 1b and 3b type, which may be one of the causes of 1B subtype which is easy to be chronic infection and is one of the causes of rapid pathogenicity.
The course of disease and ALT 6. hepatitis C, there is no significant correlation between AST and liver biochemical indexes among different genotypes of ALT, no significant differences in the level of AST, but the HCV virus load type 1b patients was significantly higher than that of patients with other genotypes, with AST, ALT and other biochemical indexes of liver function compared to HCV viral load is more suitable for hepatitis C the course evaluation index.
Early diagnosis of hepatitis C is an important means to control the spread of HCV, the detection method of HCV core protein and antibody response to HCV infection based on the "window period" diagnosis is of great value, the expression of Core protein is a key step to establish this method. In this study HCV Core gene fragments were cloned into the pET 100 / D-TopoVector the expression of BL21Chemically in Competent, Ecoli, and SDS-PAGE electrophoresis, found that the cloned Core gene was correct, and can be induced to express the protein of the expected size (about 23KD), in the 2.5 hours of culturing, can obtain the maximum expression.
In summary, the study of HCV genotypes in patients with hepatitis C in Yunnan city of Kunming province distribution are studied systematically, the results will help to clarify molecular epidemiological characteristics of HCV in Yunnan Province, tracing the spread of the HCV virus source, analysis of HCV virus evolution characteristics, at the same time for the clinical diagnosis and treatment of patients with hepatitis C in our province. Drug and vaccine development and local offers the basis of.HCV Core protein expression construct will lay the foundation for the preparation of HCV antibody diagnostic reagents with high specificity and sensitivity.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R181.3;R512.6

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