丁型肝炎病毒穩(wěn)定轉(zhuǎn)染肝癌細(xì)胞系的建立
發(fā)布時(shí)間：2018-09-11 17:48
【摘要】：丁型肝炎病毒(hepatitis D virus,HDV)是一種類(lèi)病毒樣核糖核酸(ribonucleic acid,RNA)病毒,這種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)用于病毒的組裝、釋放和感染。與單獨(dú)的HBV感染相比,合并HDV感染后會(huì)加速肝硬化的程度,會(huì)增加暴發(fā)性肝炎和肝細(xì)胞癌的發(fā)生率。但是目前關(guān)于HDV的研究還是相對(duì)缺乏,因此加強(qiáng)對(duì)HDV的進(jìn)一步研究是非常必要的。目前丁型肝炎病毒的獲取方式有兩種:一種是從丁型肝炎患者的血清中直接提取HDV,但我國(guó)丁型肝炎的患者比較少見(jiàn),若想依賴(lài)從血清中直接提取HDV的這種途徑不現(xiàn)實(shí);另外一種是基于質(zhì)粒瞬時(shí)轉(zhuǎn)染HuH-7等細(xì)胞系的HDV包裝方法,但是當(dāng)研究需要大量病毒時(shí)工作量會(huì)明顯增大,而且實(shí)驗(yàn)批次之間的可重復(fù)性也較差。最近廈門(mén)大學(xué)發(fā)表了基于腺病毒的HDV病毒包裝新方法,解決了一定問(wèn)題。但到目前為止尚沒(méi)有HDV的穩(wěn)定包裝細(xì)胞系用于HDV的大規(guī)模生產(chǎn)和抗病毒藥物評(píng)價(jià)。HDV是HBV的衛(wèi)星病毒,為HBV感染的研究提供了替代工具。本研究欲構(gòu)建HDV復(fù)制包裝轉(zhuǎn)座子載體并嘗試轉(zhuǎn)染不同的肝癌細(xì)胞系,驗(yàn)證各項(xiàng)HDV相關(guān)復(fù)制感染指標(biāo),最終篩選出具有高包裝效率和高感染性的HDV單克隆穩(wěn)定轉(zhuǎn)染細(xì)胞系。本研究首先采用分子克隆方法構(gòu)建出含HDV復(fù)制子和HBsAg表達(dá)框的piggyBac(PB)轉(zhuǎn)座子載體并進(jìn)行了功能評(píng)價(jià)。將構(gòu)建好的載體瞬時(shí)轉(zhuǎn)染Hu H-7細(xì)胞,通過(guò)檢測(cè)細(xì)胞上清HDV RNA和HBsAg的含量初步確定載體的功能完好,相關(guān)功能基因得到有效表達(dá),并通過(guò)濃縮細(xì)胞上清病毒后感染HBV受體鈉離子/�；悄懰峁厕D(zhuǎn)運(yùn)蛋白(Na+/taurocholate Cotransporting Polypeptide,NTCP)的穩(wěn)定轉(zhuǎn)染細(xì)胞系HepG2.N9確定了細(xì)胞上清含有感染性HDV顆粒。課題組成員應(yīng)用該載體和其他HDV復(fù)制包裝載體系統(tǒng)轉(zhuǎn)染了多個(gè)人來(lái)源肝癌細(xì)胞系,通過(guò)應(yīng)用嘌呤霉素快速大規(guī)模篩選最終獲得一株HDV的高包裝效率細(xì)胞系K7。本研究進(jìn)一步通過(guò)化學(xué)發(fā)光法檢測(cè)細(xì)胞上清HBV表面抗原的含量,ELISA檢測(cè)細(xì)胞上清HBV preS1的水平,以及定量PCR檢測(cè)細(xì)胞上清HDV病毒滴度,利用HBV受體NTCP穩(wěn)定轉(zhuǎn)染細(xì)胞系HepG2.N9評(píng)價(jià)了細(xì)胞上清HDV病毒體外感染性,結(jié)果表明本研究成功建立了支持HDV復(fù)制、包裝、分泌的人源肝癌細(xì)胞系K7,其細(xì)胞上清可檢測(cè)到大量的HBsAg并可檢測(cè)到HBV preS1的存在,細(xì)胞上清HDV滴度達(dá)到1E+08GE/ml(GE,genome equivalents)以上,并且證明本細(xì)胞所分泌的HDV病毒體外具有較好的感染性。本實(shí)驗(yàn)通過(guò)構(gòu)建含有HDV復(fù)制子和HBsAg表達(dá)框的HDV表達(dá)載體,嘗試篩選不同來(lái)源的人肝癌細(xì)胞系,最終成功篩選出了具有較高包裝效率和感染性的HDV包裝分泌單克隆細(xì)胞系K7,解決了HDV大規(guī)模包裝生產(chǎn)的難題,并為大規(guī)模篩選潛在抗丁型肝炎分子藥物提供了穩(wěn)定轉(zhuǎn)染細(xì)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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R373.21
本文編號(hào)：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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R373.21
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