本文選題：乙型肝炎病毒 + 熒光素酶　； 參考：《河北醫(yī)科大學(xué)》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(yán)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(xiǎn)因素。加強(qiáng)對(duì)HBV分子生物學(xué)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(shí)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(dá)熒光素酶的重組HBV復(fù)制細(xì)胞系及HBV易感細(xì)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(dá)熒光素酶的重組HBV復(fù)制細(xì)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(dá)熒光素酶標(biāo)簽的重組HBV,并且在輔助質(zhì)粒存在時(shí)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(dá)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(dá)熒光素酶sec Nluc,構(gòu)建帶有Sec Nluc熒光素酶標(biāo)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(dá)潮霉素抗性的重組HBV質(zhì)粒p TRE-HBV-S-NLuc與表達(dá)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(xì)胞系Hep G2-TA2-7細(xì)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(dá)熒光素酶的HBV復(fù)制細(xì)胞克隆。4通過熒光定量PCR檢測(cè)技術(shù)檢測(cè)各個(gè)細(xì)胞克隆上清液中的HBV DNA表達(dá)水平;同時(shí),利用Nano-Glo Luciferase Assay System試劑盒檢測(cè)這些細(xì)胞克隆上清液中熒光素酶量的表達(dá)水平。篩選出HBV DNA表達(dá)水平以及熒光素酶表達(dá)水平均較高的細(xì)胞克隆進(jìn)行進(jìn)一步的相應(yīng)檢測(cè)。5通過Native Western Blot檢測(cè)篩選出的細(xì)胞克隆的HBV核心蛋白顆粒的形成。6通過Southern blot檢測(cè)篩選出的細(xì)胞克隆中HBV DNA的形成。7通過透射電鏡觀察細(xì)胞內(nèi)病毒顆粒的形成。結(jié)果:1表達(dá)潮霉素抗性的重組HBV質(zhì)粒p TRE-HBV-S-NLuc與表達(dá)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(xì)胞系Hep G2-TA2-7細(xì)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(gè)細(xì)胞克隆繼續(xù)擴(kuò)大培養(yǎng),分別編號(hào)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(cè)了各個(gè)細(xì)胞克隆上清液中的HBV DNA表達(dá)水平以及熒光素酶的表達(dá)水平。經(jīng)過分析比較,最終篩選出HBV DNA表達(dá)水平及熒光素酶的表達(dá)量均較高的6個(gè)細(xì)胞克隆進(jìn)一步分析,分別為HBV-SNLuc-(1,12,13,24,35,36)。2經(jīng)Native Western Blot檢測(cè),可觀察到6個(gè)細(xì)胞克隆以及Hep G2.117細(xì)胞均有核心蛋白顆粒形成,結(jié)果證實(shí)HBV-SNluc細(xì)胞可以產(chǎn)生完整的核心蛋白顆粒。3經(jīng)Southern blot檢測(cè),可觀察到6個(gè)細(xì)胞克隆以及Hep G2.117細(xì)胞均有疏松環(huán)狀、雙鏈及單鏈HBV DNA的形成,結(jié)果證實(shí)HBV-SNluc細(xì)胞能夠產(chǎn)生HBV的復(fù)制中間體,并有較強(qiáng)的復(fù)制能力。4經(jīng)過上述檢測(cè),確定HBV-SNLuc-35為最佳細(xì)胞株,不僅分泌較高的HBV顆粒,也表達(dá)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(xì)胞克隆能夠產(chǎn)生與對(duì)照組Hep G2.117細(xì)胞相似的病毒顆粒。結(jié)論:利用Hep G2細(xì)胞成功構(gòu)建了穩(wěn)定表達(dá)熒光素酶的并具有HBV復(fù)制的細(xì)胞克隆,經(jīng)系列研究,篩選出一株高效表達(dá)熒光素酶和分泌重組HBV顆粒的細(xì)胞系HBV-SNLuc-35。第二部分QSG-7701細(xì)胞穩(wěn)定表達(dá)NTCP制備HBV易感細(xì)胞系的研究目的:鑒于QSG-7701細(xì)胞能夠更好的支持HBV的復(fù)制,本研究應(yīng)用QSG-7701細(xì)胞構(gòu)建穩(wěn)定表達(dá)鈉離子-牛磺膽酸共轉(zhuǎn)運(yùn)多肽(sodium taurocholate co-transporting polypeptide,NTCP)的QSG-7701的HBV易感細(xì)胞模型。方法:1在含有白蛋白啟動(dòng)子的質(zhì)粒p Alb上表達(dá)帶有Flag標(biāo)簽的NTCP基因,再插入一個(gè)潮霉素抗性基因表達(dá)盒,最終構(gòu)建了潮霉素抗性的以白蛋白啟動(dòng)子驅(qū)動(dòng)表達(dá)NTCP的質(zhì)粒pAlbHyg-NTCP-Flag。2表達(dá)NTCP的質(zhì)粒pAlbHyg-NTCP-Flag轉(zhuǎn)染肝癌細(xì)胞系QSG-7701細(xì)胞。經(jīng)潮霉素加壓篩選穩(wěn)定表達(dá)NTCP的細(xì)胞系QSG-7701NTCP,通過免疫熒光檢測(cè)觀察NTCP在細(xì)胞膜表面的表達(dá),挑選NTCP表達(dá)較強(qiáng)的細(xì)胞克隆。3復(fù)蘇本實(shí)驗(yàn)曾經(jīng)構(gòu)建的穩(wěn)定表達(dá)TC-FIAs H熒光標(biāo)記的HBV復(fù)制型細(xì)胞系TC-3-1,利用上清中濃縮的重組HBV感染QSG-7701NTCP細(xì)胞系以及對(duì)照組293T細(xì)胞、QSG-7701細(xì)胞,經(jīng)過雙砷熒光染料標(biāo)記以及染核標(biāo)記觀察雙砷熒光的分布情況。4利用表達(dá)熒光素酶的重組HBV感染穩(wěn)定表達(dá)NTCP的細(xì)胞系,通過熒光素酶的檢測(cè),觀察NTCP對(duì)HBV的易感性。結(jié)果:1 pAlbHyg-NTCP-Flag質(zhì)粒轉(zhuǎn)染QSG-7701細(xì)胞,經(jīng)潮霉素加壓篩選,選擇12個(gè)細(xì)胞克隆繼續(xù)擴(kuò)大培養(yǎng)。通過免疫熒光檢測(cè)細(xì)胞膜上NTCP的表達(dá),篩選出表達(dá)量較高的4個(gè)細(xì)胞克隆,分別命名為QSG-7701NTCP1~4其中QSG-7701NTCP2表達(dá)量最高,而未轉(zhuǎn)染NTCP的對(duì)照組QSG-7701只能看到微弱的熒光。2制備TC-FIAs H熒光標(biāo)記的重組HBV,分別感染QSG-7701NTCP2、293T以及QSG-7701細(xì)胞系,6小時(shí)后通過雙砷熒光染料標(biāo)記以及染核標(biāo)記可以看到QSG-7701NTCP能夠在細(xì)胞膜上有較強(qiáng)的熒光表達(dá),QSG-7701只能觀察到微弱的熒光,而293T細(xì)胞未觀察到熒光,證實(shí)了HBV與肝細(xì)胞膜上NTCP的結(jié)合。3利用HBV-SNLuc-35細(xì)胞上清中濃縮的病毒感染QSG-7701NTCP1~4及QSG-7701細(xì)胞系,經(jīng)過熒光素酶的檢測(cè),可以觀察到在QSG-7701NTCP1~4細(xì)胞培養(yǎng)上清液中熒光素酶的表達(dá)水平從第4天開始升高,并持續(xù)4-5天,均高于在QSG-7701細(xì)胞系中的表達(dá),其中QSG-7701NTCP4最高,從而證實(shí)了QSG-7701NTCP細(xì)胞系對(duì)HBV的易感性。結(jié)論:通過QSG-7701細(xì)胞系穩(wěn)定表達(dá)乙肝病毒受體NTCP,經(jīng)過感染性實(shí)驗(yàn)研究,篩選出一株對(duì)HBV具有較高易感性的細(xì)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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.62

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 梁聲強(qiáng);徐忠玉;胡桂華;許小華;鄒鵬程;;熒光素酶標(biāo)記的HBV細(xì)胞模型的建立[J];現(xiàn)代免疫學(xué);2014年05期

2 陳江燕;黃榮;陶穎;黃媛;羅英英;黃愛龍;胡接力;;乙肝病毒核心蛋白釘突部位基因工程改造對(duì)其功能的影響[J];生物工程學(xué)報(bào);2013年11期

3 陳瑤;丁雯;牟霞;胡妮婭;李明;吳英松;;人WFDC2啟動(dòng)子熒光素酶報(bào)告基因載體的構(gòu)建及其在卵巢癌HO8910細(xì)胞中的活性鑒定[J];中國婦幼保健;2011年25期

4 ;Antiviral Activity of Recombinant Cyanovirin-N against HSV-1[J];Virologica Sinica;2010年06期

5 姚冰;王莉;柴春彥;劉海峰;劉曉芳;李鋒;劉國艷;;基于熒光素酶發(fā)光體系測(cè)試飲用水中農(nóng)藥的綜合毒性[J];環(huán)境監(jiān)測(cè)管理與技術(shù);2010年01期

6 李艷;張海燕;王U，

本文編號(hào)：1879730