本文關(guān)鍵詞： 丙型肝炎病毒 基因型 基因亞型 5’NCR-C區(qū) 核心蛋白 診斷 檢測　出處：《昆明理工大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 丙型肝炎病毒(hepatitis C virus，HCV)呈全球分布，自然感染率約為3％，即有1.7～2.0億人為丙型肝炎感染者。近年來，HCV在部分地區(qū)的流行呈增長態(tài)勢，我國的HCV估計(jì)感染率為2.5-4.9％，感染總?cè)藬?shù)超過5000萬人。HCV一經(jīng)感染，大部分感染者將發(fā)展成慢性丙型肝炎，并有較大比例的感染者最終發(fā)展成肝硬化、肝癌。由于缺乏可以治愈的藥物和疫苗，對丙型肝炎尚不能有效的治療與預(yù)防，丙型肝炎已經(jīng)成為繼艾滋病之后的又一大嚴(yán)重危害人類健康的傳染病。 由于HCV的依賴RNA的RNA聚合酶(RdRp)缺乏校對功能，導(dǎo)致HCV基因突變頻率極高。根據(jù)HCV基因組核苷酸序列的同源性，HCV可被分為6個(gè)基因型，70多個(gè)亞型，以及眾多的準(zhǔn)種。HCV基因型分布呈現(xiàn)明顯的區(qū)域差異；丙型肝炎患者的臨床癥狀、病程進(jìn)展，及藥物治療效果均與基因型密切相關(guān)。HCV基因型的研究有助于明確HCV流行的危險(xiǎn)因素與傳播途徑，了解病毒的變異、進(jìn)化特點(diǎn)，并為地方性疫苗的研制，藥物的研究與開發(fā)，患者臨床觀察與治療措施的制定提供流行病學(xué)依據(jù)。對HCV感染的早期診斷是控制HCV傳播的重要手段，其中HCV核心抗原的檢測對處于HCV感染“窗口期”的個(gè)體檢測有很大價(jià)值。 本研究經(jīng)對采自云南省昆明市42例非吸毒所致丙型肝炎患者樣品，和14例靜脈吸毒所致HCV／HIV共感染者的樣品，進(jìn)行了血漿病毒核酸提取，HCV 5’NCR-C區(qū)基因的核苷酸序列測定與分析。發(fā)現(xiàn)： 1．本研究建立的基于5’NCR-C區(qū)的基因分型方法可以將所有待測樣本有效分型，彌補(bǔ)了5’NCR區(qū)在基因分型方法不能區(qū)分1型和6型固有缺陷。 2．昆明市非靜脈吸毒所致丙型肝炎病人中，1b亞型仍為主要流行型，其次為3型，2a亞型也有一定比例的流行，同時(shí)發(fā)現(xiàn)有6k和6n亞型，這與HCV在我國其它省份普通人群中1b／2a為主的基因型分布明顯不同。 3．昆明市靜脈吸毒人群中HCV感染者中以1b和3為主，同時(shí)發(fā)現(xiàn)有6n，沒有發(fā)現(xiàn)2a亞型，此種HCV基因型分布特點(diǎn)與東南亞國家更為相似。 4．本研究中普通人群1b型的組成更為復(fù)雜，可被分為五個(gè)組，6例靜脈吸毒1b型樣品則全部歸入第二個(gè)組中，證明昆明市普通人群HCV傳入呈多途徑方式，但對靜脈吸毒人群則以單途徑方式傳入。 5．HCV C區(qū)HLAA2型CTL抗原表位在1b與3b型之間有明顯差異，可能是1b亞型易于慢性感染并快速致病原因之一。 6．丙型肝炎病程與ALT、AST等肝功生化指標(biāo)之間無顯著相關(guān)性，不同基因型間ALT、AST水平無明顯差異，但1b型患者HCV病毒載量顯著高于其它基因型患者，與AST，ALT等肝功生化指標(biāo)相比較，HCV病毒載量更適合作為丙型肝炎病程評價(jià)指標(biāo)。 對丙型肝炎早期診斷是控制HCV傳播的重要手段，其中基于HCV核心蛋白及相關(guān)抗體反應(yīng)的檢測方法對HCV感染者“窗口期”診斷有很大價(jià)值，Core蛋白表達(dá)是建立此方法的核心步驟。本研究將HCV Core基因擴(kuò)增產(chǎn)物克隆到pET 100／D-TopoVector，在BL21Chemically Competent Ecoli中表達(dá)，進(jìn)而用SDS-PAGE電泳檢測，發(fā)現(xiàn)：經(jīng)克隆獲得了正確的Core基因，并可誘導(dǎo)表達(dá)出預(yù)期大小的蛋白分子(約23KD)，在誘導(dǎo)培養(yǎng)2.5小時(shí)，可獲得最大表達(dá)量。 綜上所述，本研究對云南省昆明市丙型肝炎患者的HCV基因型分布進(jìn)行了較為系統(tǒng)的研究，所取得結(jié)果將有助于明確云南省HCV分子流行病學(xué)特點(diǎn)、追溯HCV病毒傳播來源，分析HCV病毒進(jìn)化特點(diǎn)，同時(shí)為我省丙型肝炎患者病人臨床診斷與治療，及進(jìn)行藥物研發(fā)合地方性疫苗研制提供前期研究基礎(chǔ)。HCV Core蛋白表達(dá)構(gòu)建將為研制高特異性和敏感性的HCV抗體診斷試劑奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R181.3;R512.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 來家騏,卜建朝;丙型肝炎病毒基因變異與臨床[J];河北醫(yī)藥;2001年05期

2 黃海燕,孟祥偉;丙型肝炎基因分型[J];吉林醫(yī)學(xué);2003年06期

3 陳佩蘭,李琳,田惠英,陳良標(biāo);丙型肝炎患者外周血單個(gè)核細(xì)胞HCV和Fas抗原檢測[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);2000年03期

4 韓亞萍,劉婷,李軍,張永祥,周雄偉,鄧志龍,黃祖瑚;PCR-ELISA法檢測HCV-RNA的臨床應(yīng)用[J];現(xiàn)代檢驗(yàn)醫(yī)學(xué)雜志;2002年03期

5 趙繼義,劉雪梅,郭薇媛,高磊,鐘照華,谷鴻喜;RT套式PCR檢測血漿HCV RNA及與抗HCV檢測的比較[J];微生物學(xué)雜志;2001年04期

6 金丹,張淑芳,李紅花,魏成淑,李英信,李玉雨,孟繁平;吉林省延邊地區(qū)丙型肝炎病毒基因型分布[J];微生物學(xué)雜志;2001年04期

7 汪濤,郭華章,王海濤,金伯泉;丙型肝炎特異性細(xì)胞毒性T細(xì)胞及其免疫應(yīng)答的研究進(jìn)展[J];細(xì)胞與分子免疫學(xué)雜志;1998年04期

8 謝立,吳曉東;丙型肝炎病毒檢測方法的研究進(jìn)展及其臨床意義[J];世界華人消化雜志;2005年07期

9 楊東亮，郝連杰;我國丙型肝炎病毒基因變異及分型研究現(xiàn)狀[J];中華傳染病雜志;1996年01期

10 邢文革,石向東,陳光增,鄭懷競;國產(chǎn)丙型肝炎病毒抗體酶免檢測試劑盒的質(zhì)量評價(jià)[J];中華肝臟病雜志;2001年05期

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