丁型肝炎病毒稳定转染肝癌细胞系的建立
发布时间：2018-09-11 17:48
【摘要】：丁型肝炎病毒(hepatitis D virus,HDV)是一种类病毒样核糖核酸(ribonucleic acid,RNA)病毒,这种缺陷型病毒需要乙型肝炎病毒(hepatitis B virus,HBV)提供乙型肝炎病毒表面抗原(Hepatitis B virus surface antigen,HBsAg)用于病毒的组装、释放和感染。与单独的HBV感染相比,合并HDV感染后会加速肝硬化的程度,会增加暴发性肝炎和肝细胞癌的发生率。但是目前关于HDV的研究还是相对缺乏,因此加强对HDV的进一步研究是非常必要的。目前丁型肝炎病毒的获取方式有两种:一种是从丁型肝炎患者的血清中直接提取HDV,但我国丁型肝炎的患者比较少见,若想依赖从血清中直接提取HDV的这种途径不现实;另外一种是基于质粒瞬时转染HuH-7等细胞系的HDV包装方法,但是当研究需要大量病毒时工作量会明显增大,而且实验批次之间的可重复性也较差。最近厦门大学发表了基于腺病毒的HDV病毒包装新方法,解决了一定问题。但到目前为止尚没有HDV的稳定包装细胞系用于HDV的大规模生产和抗病毒药物评价。HDV是HBV的卫星病毒,为HBV感染的研究提供了替代工具。本研究欲构建HDV复制包装转座子载体并尝试转染不同的肝癌细胞系,验证各项HDV相关复制感染指标,最终筛选出具有高包装效率和高感染性的HDV单克隆稳定转染细胞系。本研究首先采用分子克隆方法构建出含HDV复制子和HBsAg表达框的piggyBac(PB)转座子载体并进行了功能评价。将构建好的载体瞬时转染Hu H-7细胞,通过检测细胞上清HDV RNA和HBsAg的含量初步确定载体的功能完好,相关功能基因得到有效表达,并通过浓缩细胞上清病毒后感染HBV受体钠离子/牛磺胆酸共转运蛋白(Na+/taurocholate Cotransporting Polypeptide,NTCP)的稳定转染细胞系HepG2.N9确定了细胞上清含有感染性HDV颗粒。课题组成员应用该载体和其他HDV复制包装载体系统转染了多个人来源肝癌细胞系,通过应用嘌呤霉素快速大规模筛选最终获得一株HDV的高包装效率细胞系K7。本研究进一步通过化学发光法检测细胞上清HBV表面抗原的含量,ELISA检测细胞上清HBV preS1的水平,以及定量PCR检测细胞上清HDV病毒滴度,利用HBV受体NTCP稳定转染细胞系HepG2.N9评价了细胞上清HDV病毒体外感染性,结果表明本研究成功建立了支持HDV复制、包装、分泌的人源肝癌细胞系K7,其细胞上清可检测到大量的HBsAg并可检测到HBV preS1的存在,细胞上清HDV滴度达到1E+08GE/ml(GE,genome equivalents)以上,并且证明本细胞所分泌的HDV病毒体外具有较好的感染性。本实验通过构建含有HDV复制子和HBsAg表达框的HDV表达载体,尝试筛选不同来源的人肝癌细胞系,最终成功筛选出了具有较高包装效率和感染性的HDV包装分泌单克隆细胞系K7,解决了HDV大规模包装生产的难题,并为大规模筛选潜在抗丁型肝炎分子药物提供了稳定转染细胞模型。
[Abstract]:Hepatitis D virus (hepatitis D virus,HDV) is a kind of virus-like ribonucleic acid (ribonucleic acid,RNA) virus, which requires hepatitis B virus (hepatitis B virus,HBV) to provide hepatitis B virus surface antigen (Hepatitis B virus surface antigen,HBsAg) for assembly, release and infection. Compared with HBV infection alone, HDV infection accelerates the degree of cirrhosis and increases the incidence of fulminant hepatitis and hepatocellular carcinoma. However, the current research on HDV is still relatively scarce, so it is necessary to strengthen the further study of HDV. At present, there are two ways to obtain hepatitis D virus: one is to extract HDV, directly from the serum of hepatitis D patients, but it is rare for patients with hepatitis D in China to rely on the direct extraction of HDV from the serum. The other is the HDV packaging method based on the transient transfection of plasmid into HuH-7 and other cell lines, but the workload will be increased when the research needs a large number of viruses, and the repeatability between the experimental batches is also poor. Xiamen University recently published a new method of HDV virus packaging based on adenovirus, which solves some problems. Up to now, however, there is no stable packaging cell line of HDV for mass production of HDV and evaluation of antiviral drugs. HDV is a satellite virus of HBV, which provides an alternative tool for the study of HBV infection. In this study, we constructed HDV replication packaging transposon vector and tried to transfect different hepatoma cell lines to verify the various HDV related replication and infection indexes. Finally, we screened out HDV monoclonal stable transfection cell lines with high packaging efficiency and high infectivity. In this study, the piggyBac (PB) transposon vector containing HDV replicon and HBsAg expression frame was constructed by molecular cloning and its function was evaluated. The constructed vector was transiently transfected into Hu H-7 cells. By detecting the content of HDV RNA and HBsAg in the supernatant, it was confirmed that the function of the vector was intact and the related functional genes were effectively expressed. The supernatant of HBV receptor sodium ion / taurocholate Cotransporting Polypeptide,NTCP (Na / taurocholate Cotransporting Polypeptide,NTCP) was confirmed to contain infectious HDV particles in the supernatant of the cells by the stable transfection of Na / taurocholate Cotransporting Polypeptide,NTCP. Several human hepatoma cell lines were transfected with this vector and other HDV replication and packaging vectors. A high packaging efficiency cell line K7 of HDV was obtained by rapid and large-scale screening of purine mycin. In this study, the content of HBV surface antigen in cell supernatant was detected by chemiluminescence assay. The level of HBV preS1 in supernatant was detected by Elisa, and the titer of HDV virus in supernatant was detected by quantitative PCR. The infection of supernatant HDV virus in vitro was evaluated by stably transfection of HBV receptor NTCP into HepG2.N9 cell line. The results showed that the replication and packaging of HDV were successfully established in this study. A large number of HBsAg and HBV preS1 were detected in the supernatant of the secreted human hepatoma cell line K7. The HDV titer of the supernatant of the cell reached 1 E 08GE/ml (GE,genome equivalents), which proved that the HDV virus secreted by the cell had a good infection in vitro. In this study, HDV expression vectors containing HDV replicon and HBsAg expression frame were constructed to screen human hepatoma cell lines from different sources. Finally, the monoclonal cell line K7, which has higher packaging efficiency and infectivity, was successfully screened out, which solved the problem of HDV packaging production on a large scale. It also provides a stable transfection cell model for screening potential hepatitis D molecular drugs in large scale.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.21
本文编号：2237411
[Abstract]:Hepatitis D virus (hepatitis D virus,HDV) is a kind of virus-like ribonucleic acid (ribonucleic acid,RNA) virus, which requires hepatitis B virus (hepatitis B virus,HBV) to provide hepatitis B virus surface antigen (Hepatitis B virus surface antigen,HBsAg) for assembly, release and infection. Compared with HBV infection alone, HDV infection accelerates the degree of cirrhosis and increases the incidence of fulminant hepatitis and hepatocellular carcinoma. However, the current research on HDV is still relatively scarce, so it is necessary to strengthen the further study of HDV. At present, there are two ways to obtain hepatitis D virus: one is to extract HDV, directly from the serum of hepatitis D patients, but it is rare for patients with hepatitis D in China to rely on the direct extraction of HDV from the serum. The other is the HDV packaging method based on the transient transfection of plasmid into HuH-7 and other cell lines, but the workload will be increased when the research needs a large number of viruses, and the repeatability between the experimental batches is also poor. Xiamen University recently published a new method of HDV virus packaging based on adenovirus, which solves some problems. Up to now, however, there is no stable packaging cell line of HDV for mass production of HDV and evaluation of antiviral drugs. HDV is a satellite virus of HBV, which provides an alternative tool for the study of HBV infection. In this study, we constructed HDV replication packaging transposon vector and tried to transfect different hepatoma cell lines to verify the various HDV related replication and infection indexes. Finally, we screened out HDV monoclonal stable transfection cell lines with high packaging efficiency and high infectivity. In this study, the piggyBac (PB) transposon vector containing HDV replicon and HBsAg expression frame was constructed by molecular cloning and its function was evaluated. The constructed vector was transiently transfected into Hu H-7 cells. By detecting the content of HDV RNA and HBsAg in the supernatant, it was confirmed that the function of the vector was intact and the related functional genes were effectively expressed. The supernatant of HBV receptor sodium ion / taurocholate Cotransporting Polypeptide,NTCP (Na / taurocholate Cotransporting Polypeptide,NTCP) was confirmed to contain infectious HDV particles in the supernatant of the cells by the stable transfection of Na / taurocholate Cotransporting Polypeptide,NTCP. Several human hepatoma cell lines were transfected with this vector and other HDV replication and packaging vectors. A high packaging efficiency cell line K7 of HDV was obtained by rapid and large-scale screening of purine mycin. In this study, the content of HBV surface antigen in cell supernatant was detected by chemiluminescence assay. The level of HBV preS1 in supernatant was detected by Elisa, and the titer of HDV virus in supernatant was detected by quantitative PCR. The infection of supernatant HDV virus in vitro was evaluated by stably transfection of HBV receptor NTCP into HepG2.N9 cell line. The results showed that the replication and packaging of HDV were successfully established in this study. A large number of HBsAg and HBV preS1 were detected in the supernatant of the secreted human hepatoma cell line K7. The HDV titer of the supernatant of the cell reached 1 E 08GE/ml (GE,genome equivalents), which proved that the HDV virus secreted by the cell had a good infection in vitro. In this study, HDV expression vectors containing HDV replicon and HBsAg expression frame were constructed to screen human hepatoma cell lines from different sources. Finally, the monoclonal cell line K7, which has higher packaging efficiency and infectivity, was successfully screened out, which solved the problem of HDV packaging production on a large scale. It also provides a stable transfection cell model for screening potential hepatitis D molecular drugs in large scale.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.21
【参考文献】
相关期刊论文 前10条
1 郑猛;;病毒性肝炎的预防及其疫苗应用[J];世界最新医学信息文摘;2016年30期
2 Celso Cunha;Jo?o Paulo Tavanez;Severin Gudima;;Hepatitis delta virus: A fascinating and neglected pathogen[J];World Journal of Virology;2015年04期
3 王融冰;;肝病的中医药治疗[J];临床肝胆病杂志;2015年01期
4 叶振东;;分析病毒性肝炎的中医治疗[J];中医临床研究;2014年17期
5 John M Taylor;;Host RNA circles and the origin of hepatitis delta virus[J];World Journal of Gastroenterology;2014年11期
6 吴志德;周沛林;;丁型肝炎病毒感染标志物在乙型肝炎病毒感染者中的检测研究[J];中华医院感染学杂志;2013年13期
7 许泼实;韩双印;孙长义;赵静;;乙型肝炎病毒感染者血清中丁型肝炎病毒标志物检测分析[J];中华实验和临床病毒学杂志;2012年04期
8 杨松;邢卉春;成军;;慢性丁型肝炎抗病毒治疗进展[J];中华实验和临床感染病杂志(电子版);2011年04期
9 邵惠训;;一种缺陷病毒——丁型肝炎病毒[J];首都公共卫生;2011年03期
10 卢学新;伊瑶;毕胜利;;丁型肝炎的分子生物学特征及研究进展[J];中华实验和临床病毒学杂志;2013年03期
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