稳定表达荧光素酶的重组HBV复制细胞系及HBV易感细胞系的构建

发布时间：2018-05-12 18:48

本文选题：乙型肝炎病毒 + 荧光素酶　；参考：《河北医科大学》2017年硕士论文

【摘要】：HBV引起的慢性乙型病毒性肝炎及其相关疾病严重危害人类健康。HBV持续感染是发展成肝硬化、肝癌的重要危险因素。加强对HBV分子生物学的研究,为指导临床治疗和新药的发现提供重要的依据,同时将有助于降低HBV的发病率和死亡率。本研究中,我们通过构建稳定表达荧光素酶的重组HBV复制细胞系及HBV易感细胞系,为HBV感染模型的研究及受体抑制剂的研究提供了有利工具。第一部分稳定表达荧光素酶的重组HBV复制细胞系的构建目的:将乙型肝炎病毒(Hepatitis B virus,HBV)改造成可以携带表达荧光素酶标签的重组HBV,并且在辅助质粒存在时仍有复制能力并能形成完整的HBV颗粒。方法:1应用PCR及分子克隆技术,利用表达野生型HBV的p TRE-HBV质粒,在HBV S区HBs Ag的Xho I-Bsr GI区段表达荧光素酶sec Nluc,构建带有Sec Nluc荧光素酶标签的p TRE-HBV-S-NLuc的重组HBV质粒。2构建具有杀稻瘟菌素抗性的HBV辅助质粒pc DNA3.1(-)Bsd-CH3142。3表达潮霉素抗性的重组HBV质粒p TRE-HBV-S-NLuc与表达杀稻瘟菌素抗性的HBV辅助质粒pc DNA3.1(-)Bsd-CH3142以1:1的比例共转染肝癌细胞系Hep G2-TA2-7细胞。经新霉素、潮霉素和杀稻瘟菌素共同加压筛选稳定表达荧光素酶的HBV复制细胞克隆。4通过荧光定量PCR检测技术检测各个细胞克隆上清液中的HBV DNA表达水平;同时,利用Nano-Glo Luciferase Assay System试剂盒检测这些细胞克隆上清液中荧光素酶量的表达水平。筛选出HBV DNA表达水平以及荧光素酶表达水平均较高的细胞克隆进行进一步的相应检测。5通过Native Western Blot检测筛选出的细胞克隆的HBV核心蛋白颗粒的形成。6通过Southern blot检测筛选出的细胞克隆中HBV DNA的形成。7通过透射电镜观察细胞内病毒颗粒的形成。结果:1表达潮霉素抗性的重组HBV质粒p TRE-HBV-S-NLuc与表达杀稻瘟菌素抗性的HBV辅助质粒pc DNA3.1(-)Bsd-CH3142以1:1的比例共转染肝癌细胞系Hep G2-TA2-7细胞,经新霉素、潮霉素和杀稻瘟菌素三种抗生素共同加压筛选,挑选36个细胞克隆继续扩大培养,分别编号HBV-SNLuc1~36,并应用荧光定量PCR方法和Nano-Glo Luciferase Assay System试剂盒分别检测了各个细胞克隆上清液中的HBV DNA表达水平以及荧光素酶的表达水平。经过分析比较,最终筛选出HBV DNA表达水平及荧光素酶的表达量均较高的6个细胞克隆进一步分析,分别为HBV-SNLuc-(1,12,13,24,35,36)。2经Native Western Blot检测,可观察到6个细胞克隆以及Hep G2.117细胞均有核心蛋白颗粒形成,结果证实HBV-SNluc细胞可以产生完整的核心蛋白颗粒。3经Southern blot检测,可观察到6个细胞克隆以及Hep G2.117细胞均有疏松环状、双链及单链HBV DNA的形成,结果证实HBV-SNluc细胞能够产生HBV的复制中间体,并有较强的复制能力。4经过上述检测,确定HBV-SNLuc-35为最佳细胞株,不仅分泌较高的HBV颗粒,也表达较高水平的荧光素酶。5通过透射电镜观察,可以看到HBV-SNLuc-35细胞克隆能够产生与对照组Hep G2.117细胞相似的病毒颗粒。结论:利用Hep G2细胞成功构建了稳定表达荧光素酶的并具有HBV复制的细胞克隆,经系列研究,筛选出一株高效表达荧光素酶和分泌重组HBV颗粒的细胞系HBV-SNLuc-35。第二部分QSG-7701细胞稳定表达NTCP制备HBV易感细胞系的研究目的:鉴于QSG-7701细胞能够更好的支持HBV的复制,本研究应用QSG-7701细胞构建稳定表达钠离子-牛磺胆酸共转运多肽(sodium taurocholate co-transporting polypeptide,NTCP)的QSG-7701的HBV易感细胞模型。方法:1在含有白蛋白启动子的质粒p Alb上表达带有Flag标签的NTCP基因,再插入一个潮霉素抗性基因表达盒,最终构建了潮霉素抗性的以白蛋白启动子驱动表达NTCP的质粒pAlbHyg-NTCP-Flag。2表达NTCP的质粒pAlbHyg-NTCP-Flag转染肝癌细胞系QSG-7701细胞。经潮霉素加压筛选稳定表达NTCP的细胞系QSG-7701NTCP,通过免疫荧光检测观察NTCP在细胞膜表面的表达,挑选NTCP表达较强的细胞克隆。3复苏本实验曾经构建的稳定表达TC-FIAs H荧光标记的HBV复制型细胞系TC-3-1,利用上清中浓缩的重组HBV感染QSG-7701NTCP细胞系以及对照组293T细胞、QSG-7701细胞,经过双砷荧光染料标记以及染核标记观察双砷荧光的分布情况。4利用表达荧光素酶的重组HBV感染稳定表达NTCP的细胞系,通过荧光素酶的检测,观察NTCP对HBV的易感性。结果:1 pAlbHyg-NTCP-Flag质粒转染QSG-7701细胞,经潮霉素加压筛选,选择12个细胞克隆继续扩大培养。通过免疫荧光检测细胞膜上NTCP的表达,筛选出表达量较高的4个细胞克隆,分别命名为QSG-7701NTCP1~4其中QSG-7701NTCP2表达量最高,而未转染NTCP的对照组QSG-7701只能看到微弱的荧光。2制备TC-FIAs H荧光标记的重组HBV,分别感染QSG-7701NTCP2、293T以及QSG-7701细胞系,6小时后通过双砷荧光染料标记以及染核标记可以看到QSG-7701NTCP能够在细胞膜上有较强的荧光表达,QSG-7701只能观察到微弱的荧光,而293T细胞未观察到荧光,证实了HBV与肝细胞膜上NTCP的结合。3利用HBV-SNLuc-35细胞上清中浓缩的病毒感染QSG-7701NTCP1~4及QSG-7701细胞系,经过荧光素酶的检测,可以观察到在QSG-7701NTCP1~4细胞培养上清液中荧光素酶的表达水平从第4天开始升高,并持续4-5天,均高于在QSG-7701细胞系中的表达,其中QSG-7701NTCP4最高,从而证实了QSG-7701NTCP细胞系对HBV的易感性。结论:通过QSG-7701细胞系稳定表达乙肝病毒受体NTCP,经过感染性实验研究,筛选出一株对HBV具有较高易感性的细胞系QSG-7701NTCP4。
[Abstract]:Chronic hepatitis B caused by HBV and its related diseases seriously endangers human health.HBV continuous infection is an important risk factor for developing liver cirrhosis and liver cancer. Strengthening the study of HBV molecular biology provides important basis for guiding clinical treatment and discovery of new drugs, and will help to reduce the incidence and mortality of HBV at the same time. In this study, we constructed a recombinant HBV replicating cell line and a HBV susceptible cell line that stably expressed luciferase, and provided a useful tool for the study of HBV infection model and the research of receptor inhibitors. HBV) is transformed into a recombinant HBV that can carry the label of luciferase, and still has the ability to replicate and form a complete HBV particle in the presence of the auxiliary plasmid. Method: 1, using PCR and molecular cloning technology, the P TRE-HBV plasmid expressing wild type HBV was used to express luciferase and construct the band in the HBs Ag HBV S region of HBV S region. Recombinant HBV plasmid.2 with Sec Nluc luciferase label P TRE-HBV-S-NLuc construction of HBV assisted plasmid PC DNA3.1 (-) Bsd-CH3142.3 expressing hygromycin resistance of oryzicocine - Bsd-CH3142.3 Cell line Hep G2-TA2-7 cells. The HBV replicating cell clone of stable expression luciferase was screened by CO compression of neomycin, hygromycin and grisemonin. The expression of HBV DNA expression in the supernatant of each cell was detected by the fluorescence quantitative PCR detection technique. Meanwhile, the Nano-Glo Luciferase Assay System kit was used to detect these fines. The expression level of luciferase in the supernatant of cloned cells. Screening out HBV DNA expression level and high luciferase expression level of cell clones to further detect the formation of HBV core protein particles formed by.5 through the Native Western Blot detection of the formation of.6 through Southern blot detection of the cells The formation of HBV DNA in the clone was observed by transmission electron microscopy. Results: 1 the recombinant HBV plasmid P TRE-HBV-S-NLuc of hygromycin resistance and HBV assisted plasmid PC DNA3.1 (-) Bsd-CH3142 expressing the resistance to oryzicin, PC DNA3.1 (-) Bsd-CH3142, were co transfected to the hepatocellular carcinoma cell line Hep squamous cell, and neomycin, hygromycin and Hygromycin Three kinds of antibiotics were screened and 36 cell clones were selected to continue to expand culture, HBV-SNLuc1~36. The level of HBV DNA and the level of luciferase expression in the supernatant of each cell were detected by fluorescence quantitative PCR method and Nano-Glo Luciferase Assay System kit. After the analysis and comparison, 6 cells with high expression level of HBV DNA and the high expression of luciferase were further analyzed. HBV-SNLuc- (1,12,13,24,35,36).2 was detected by Native Western Blot, respectively. 6 cell clones and Hep G2.117 cells were observed to have nucleate protein particles. The results confirmed that HBV-SNluc cells could be found. The complete core protein particle.3 was detected by Southern blot. It was observed that 6 cell clones and Hep G2.117 cells had loose ring, double chain and single strand HBV DNA. The results confirmed that HBV-SNluc cells could produce HBV replication intermediates, and a strong replication energy.4 was found to be the best for HBV-SNLuc-35. The cell line, which not only secretes high HBV particles but also expresses high level luciferase.5 through transmission electron microscopy, can see that HBV-SNLuc-35 cell clones can produce virus particles similar to that of the control group Hep G2.117 cells. Conclusion: using Hep G2 cells, a successful construction of a stable expression luciferase and a HBV replicating cell gram is constructed. On the basis of a series of studies, the purpose of this study was to establish a stable expression of HBV susceptible cell lines with a stable expression of QSG-7701 cells, HBV-SNLuc-35. second QSG-7701 cells, which efficiently expressed luciferase and secreted recombinant HBV granules. The purpose of this study was to construct a stable expression of sodium from QSG-7701 cells in view of the ability of QSG-7701 cells to better support the replication of HBV. HBV susceptible cell model of QSG-7701 of sodium taurocholate co-transporting polypeptide, NTCP. Method: 1 expression of NTCP gene with Flag label on the plasmid P Alb containing albumin promoter, and then a hygromycin resistance gene expression box was inserted, and the white egg resistant to hygromycin was finally constructed. The plasmid pAlbHyg-NTCP-Flag expressing the plasmid pAlbHyg-NTCP-Flag.2 expressing the NTCP expression NTCP was transfected to the QSG-7701 cells of the hepatocellular carcinoma cell line. The cell line QSG-7701NTCP which expressed NTCP was screened by hygromycin pressure, and the surface of NTCP on the surface of the cell membrane was observed by immunofluorescence, and the NTCP expression of the cell clone.3 was selected. The HBV replicative cell line, TC-3-1, which has been constructed by the Suen experiment to express the stable expression of TC-FIAs H fluorescence, is used to infect QSG-7701NTCP cell lines in the supernatant HBV and the 293T cells of the control group, QSG-7701 cells, and the distribution of arsenic fluorescence with double arsenic fluorescent dyes and staining markers to observe the distribution of double arsenic fluorescence in.4 to express luciferase The recombinant HBV infected the cell line of NTCP steadily. Through the detection of luciferase, the susceptibility of NTCP to HBV was observed. Results: 1 pAlbHyg-NTCP-Flag plasmid transfected to QSG-7701 cells and screened by hygromycin and selected 12 cell clones to continue to expand culture. The expression of NTCP on the cell membrane was detected by immunofluorescence, and the expression was higher. The 4 cell clones, named QSG-7701NTCP1~4, were named the highest QSG-7701NTCP2 expression, while the control group that did not transfect NTCP could only see a weak fluorescent.2 for the preparation of the TC-FIAs H fluorescence labeled recombinant HBV, respectively, to infect QSG-7701NTCP2293T and QSG-7701 cell lines, and 6 small hours later through double arsenic fluorescent dye labeling and dyed nuclear markers. It is noted that QSG-7701NTCP can have strong fluorescence expression on the cell membrane, QSG-7701 can only observe the weak fluorescence, and 293T cells do not observe the fluorescence, which confirms that the combination of HBV and NTCP on the hepatic cell membrane is infected with the QSG-7701NTCP1~4 and QSG-7701 cell lines, which are concentrated in the HBV-SNLuc-35 cell supernatant, and through luciferase. It was observed that the level of luciferase expression in the supernatant of QSG-7701NTCP1~4 cell culture increased from fourth days and lasted for 4-5 days, which was higher than that in the QSG-7701 cell line. QSG-7701NTCP4 was the highest, which confirmed the susceptibility to HBV in the QSG-7701NTCP cell line. Conclusion: the expression of the QSG-7701 cell line is stable. HBV receptor NTCP has been screened for a highly susceptible cell line QSG-7701NTCP4. by HBV.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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