發(fā)布時間：2018-05-12 18:48

  本文選題：乙型肝炎病毒 + 熒光素酶��； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：HBV引起的慢性乙型病毒性肝炎及其相關(guān)疾病嚴重危害人類健康。HBV持續(xù)感染是發(fā)展成肝硬化、肝癌的重要危險因素。加強對HBV分子生物學的研究,為指導(dǎo)臨床治療和新藥的發(fā)現(xiàn)提供重要的依據(jù),同時將有助于降低HBV的發(fā)病率和死亡率。本研究中,我們通過構(gòu)建穩(wěn)定表達熒光素酶的重組HBV復(fù)制細胞系及HBV易感細胞系,為HBV感染模型的研究及受體抑制劑的研究提供了有利工具。第一部分穩(wěn)定表達熒光素酶的重組HBV復(fù)制細胞系的構(gòu)建目的:將乙型肝炎病毒(Hepatitis B virus,HBV)改造成可以攜帶表達熒光素酶標簽的重組HBV,并且在輔助質(zhì)粒存在時仍有復(fù)制能力并能形成完整的HBV顆粒。方法:1應(yīng)用PCR及分子克隆技術(shù),利用表達野生型HBV的p TRE-HBV質(zhì)粒,在HBV S區(qū)HBs Ag的Xho I-Bsr GI區(qū)段表達熒光素酶sec Nluc,構(gòu)建帶有Sec Nluc熒光素酶標簽的p TRE-HBV-S-NLuc的重組HBV質(zhì)粒。2構(gòu)建具有殺稻瘟菌素抗性的HBV輔助質(zhì)粒pc DNA3.1(-)Bsd-CH3142。3表達潮霉素抗性的重組HBV質(zhì)粒p TRE-HBV-S-NLuc與表達殺稻瘟菌素抗性的HBV輔助質(zhì)粒pc DNA3.1(-)Bsd-CH3142以1:1的比例共轉(zhuǎn)染肝癌細胞系Hep G2-TA2-7細胞。經(jīng)新霉素、潮霉素和殺稻瘟菌素共同加壓篩選穩(wěn)定表達熒光素酶的HBV復(fù)制細胞克隆。4通過熒光定量PCR檢測技術(shù)檢測各個細胞克隆上清液中的HBV DNA表達水平;同時,利用Nano-Glo Luciferase Assay System試劑盒檢測這些細胞克隆上清液中熒光素酶量的表達水平。篩選出HBV DNA表達水平以及熒光素酶表達水平均較高的細胞克隆進行進一步的相應(yīng)檢測。5通過Native Western Blot檢測篩選出的細胞克隆的HBV核心蛋白顆粒的形成。6通過Southern blot檢測篩選出的細胞克隆中HBV DNA的形成。7通過透射電鏡觀察細胞內(nèi)病毒顆粒的形成。結(jié)果:1表達潮霉素抗性的重組HBV質(zhì)粒p TRE-HBV-S-NLuc與表達殺稻瘟菌素抗性的HBV輔助質(zhì)粒pc DNA3.1(-)Bsd-CH3142以1:1的比例共轉(zhuǎn)染肝癌細胞系Hep G2-TA2-7細胞,經(jīng)新霉素、潮霉素和殺稻瘟菌素三種抗生素共同加壓篩選,挑選36個細胞克隆繼續(xù)擴大培養(yǎng),分別編號HBV-SNLuc1~36,并應(yīng)用熒光定量PCR方法和Nano-Glo Luciferase Assay System試劑盒分別檢測了各個細胞克隆上清液中的HBV DNA表達水平以及熒光素酶的表達水平。經(jīng)過分析比較,最終篩選出HBV DNA表達水平及熒光素酶的表達量均較高的6個細胞克隆進一步分析,分別為HBV-SNLuc-(1,12,13,24,35,36)。2經(jīng)Native Western Blot檢測,可觀察到6個細胞克隆以及Hep G2.117細胞均有核心蛋白顆粒形成,結(jié)果證實HBV-SNluc細胞可以產(chǎn)生完整的核心蛋白顆粒。3經(jīng)Southern blot檢測,可觀察到6個細胞克隆以及Hep G2.117細胞均有疏松環(huán)狀、雙鏈及單鏈HBV DNA的形成,結(jié)果證實HBV-SNluc細胞能夠產(chǎn)生HBV的復(fù)制中間體,并有較強的復(fù)制能力。4經(jīng)過上述檢測,確定HBV-SNLuc-35為最佳細胞株,不僅分泌較高的HBV顆粒,也表達較高水平的熒光素酶。5通過透射電鏡觀察,可以看到HBV-SNLuc-35細胞克隆能夠產(chǎn)生與對照組Hep G2.117細胞相似的病毒顆粒。結(jié)論:利用Hep G2細胞成功構(gòu)建了穩(wěn)定表達熒光素酶的并具有HBV復(fù)制的細胞克隆,經(jīng)系列研究,篩選出一株高效表達熒光素酶和分泌重組HBV顆粒的細胞系HBV-SNLuc-35。第二部分QSG-7701細胞穩(wěn)定表達NTCP制備HBV易感細胞系的研究目的:鑒于QSG-7701細胞能夠更好的支持HBV的復(fù)制,本研究應(yīng)用QSG-7701細胞構(gòu)建穩(wěn)定表達鈉離子-�；悄懰峁厕D(zhuǎn)運多肽(sodium taurocholate co-transporting polypeptide,NTCP)的QSG-7701的HBV易感細胞模型。方法:1在含有白蛋白啟動子的質(zhì)粒p Alb上表達帶有Flag標簽的NTCP基因,再插入一個潮霉素抗性基因表達盒,最終構(gòu)建了潮霉素抗性的以白蛋白啟動子驅(qū)動表達NTCP的質(zhì)粒pAlbHyg-NTCP-Flag。2表達NTCP的質(zhì)粒pAlbHyg-NTCP-Flag轉(zhuǎn)染肝癌細胞系QSG-7701細胞。經(jīng)潮霉素加壓篩選穩(wěn)定表達NTCP的細胞系QSG-7701NTCP,通過免疫熒光檢測觀察NTCP在細胞膜表面的表達,挑選NTCP表達較強的細胞克隆。3復(fù)蘇本實驗曾經(jīng)構(gòu)建的穩(wěn)定表達TC-FIAs H熒光標記的HBV復(fù)制型細胞系TC-3-1,利用上清中濃縮的重組HBV感染QSG-7701NTCP細胞系以及對照組293T細胞、QSG-7701細胞,經(jīng)過雙砷熒光染料標記以及染核標記觀察雙砷熒光的分布情況。4利用表達熒光素酶的重組HBV感染穩(wěn)定表達NTCP的細胞系,通過熒光素酶的檢測,觀察NTCP對HBV的易感性。結(jié)果:1 pAlbHyg-NTCP-Flag質(zhì)粒轉(zhuǎn)染QSG-7701細胞,經(jīng)潮霉素加壓篩選,選擇12個細胞克隆繼續(xù)擴大培養(yǎng)。通過免疫熒光檢測細胞膜上NTCP的表達,篩選出表達量較高的4個細胞克隆,分別命名為QSG-7701NTCP1~4其中QSG-7701NTCP2表達量最高,而未轉(zhuǎn)染NTCP的對照組QSG-7701只能看到微弱的熒光。2制備TC-FIAs H熒光標記的重組HBV,分別感染QSG-7701NTCP2、293T以及QSG-7701細胞系,6小時后通過雙砷熒光染料標記以及染核標記可以看到QSG-7701NTCP能夠在細胞膜上有較強的熒光表達,QSG-7701只能觀察到微弱的熒光,而293T細胞未觀察到熒光,證實了HBV與肝細胞膜上NTCP的結(jié)合。3利用HBV-SNLuc-35細胞上清中濃縮的病毒感染QSG-7701NTCP1~4及QSG-7701細胞系,經(jīng)過熒光素酶的檢測,可以觀察到在QSG-7701NTCP1~4細胞培養(yǎng)上清液中熒光素酶的表達水平從第4天開始升高,并持續(xù)4-5天,均高于在QSG-7701細胞系中的表達,其中QSG-7701NTCP4最高,從而證實了QSG-7701NTCP細胞系對HBV的易感性。結(jié)論:通過QSG-7701細胞系穩(wěn)定表達乙肝病毒受體NTCP,經(jīng)過感染性實驗研究,篩選出一株對HBV具有較高易感性的細胞系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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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本文編號：1879730