本文選題：丙型肝炎 + 疫苗　； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:丙型肝炎是由丙型肝炎病毒(Hepatitis C virus,HCV)引起的慢性肝病,是一個(gè)全球性健康問題。目前,臨床上仍無有效的治療方法。因此,開發(fā)有效的預(yù)防和治療性HCV疫苗,就成了一個(gè)亟待解決的問題。 HCV屬于黃病毒科丙型肝炎病毒屬,由于丙型肝炎病毒(hepatitis C virus,HCV)有多種基因型以及基因型的高度變異性,尤其是包膜區(qū)基因變異更大,因此按傳統(tǒng)方式研制的HCV疫苗面臨著很多困難。將編碼目的抗原的外源基因?qū)氡唤臃N者的各類型新型疫苗可望成為丙肝疫苗研發(fā)的一條新的思路。丙肝病毒核心區(qū)(C區(qū))是最為保守及穩(wěn)定的區(qū)域,有報(bào)導(dǎo)表明C抗原可誘導(dǎo)針對(duì)丙肝病毒的高水平特異性抗體應(yīng)答及細(xì)胞毒性T淋巴細(xì)胞( cytotoxic T lymphocyte, CTL )活性細(xì)胞免疫,是研制HCV DNA疫苗的主要靶抗原之一。 與質(zhì)粒載體相比,復(fù)制缺陷型腺病毒載體系統(tǒng)(replication-deficient adenovirus vector system)具有進(jìn)入宿主細(xì)胞的能力強(qiáng),宿主范圍廣,極高的轉(zhuǎn)染效率,外源基因高表達(dá)以及病毒滴度高等特點(diǎn),在疫苗載體選擇中表現(xiàn)出優(yōu)勢(shì),成為極具應(yīng)用前景的基因轉(zhuǎn)移載體。本研究中我們利用以重組復(fù)制缺陷型腺病毒為載體以HCV C抗原作為有效的靶抗原構(gòu)建了可表達(dá)HCV核心蛋白(HCV core protein)的重組腺病毒rAd-C,以探索研制丙型肝炎病毒疫苗新的途徑。 方法:1.目的基因的擴(kuò)增與鑒定以重組質(zhì)粒pEGFP-HCV/C為模板,擴(kuò)增C目的基因。將擴(kuò)增產(chǎn)物與pGEM-T載體連接,構(gòu)建質(zhì)粒pGEM-T/C。轉(zhuǎn)化DH5α大腸桿菌,提取質(zhì)粒進(jìn)行酶切鑒定及測(cè)序,篩選重組子。 2.構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.0/C將質(zhì)粒pGEM-T/C以合適的限制性內(nèi)切酶進(jìn)行雙酶切,膠回收目的片段,并與經(jīng)同樣的限制性內(nèi)切酶雙酶切的pcDNA3.0質(zhì)粒載體連接,經(jīng)轉(zhuǎn)化、篩選和酶切鑒定,構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.0/C。 3.構(gòu)建重組穿梭質(zhì)粒將質(zhì)粒pGEM-T/C以合適的限制性內(nèi)切酶進(jìn)行雙酶切,回收目的片段,將目的片段與經(jīng)合適的限制性內(nèi)切酶雙酶切的腺病毒穿梭載體pAdTrack-CMV連接。經(jīng)轉(zhuǎn)化、篩選和酶切鑒定,獲得重組穿梭質(zhì)粒pAdTrack-CMV/C。 4.重組腺病毒質(zhì)粒的構(gòu)建將質(zhì)粒用內(nèi)切酶PmeI線性化,利用AdEasy系統(tǒng),經(jīng)兩步法分別在大腸桿菌BJ-5183與骨架載體pAdEasy-1進(jìn)行同源重組,構(gòu)建重組腺病毒質(zhì)粒pAd/C。用PacI酶切鑒定,將陽性質(zhì)粒轉(zhuǎn)化入感受態(tài)細(xì)胞E.coli DH5α,以堿裂解法大量提取,并用聚乙二醇(polyethylene glycol,PEG)沉淀法進(jìn)行純化。 5.重組腺病毒的包裝與擴(kuò)增將重組腺病毒質(zhì)粒pAd/C用內(nèi)切酶Pac-I線性化,以陽離子脂質(zhì)體法轉(zhuǎn)染HEK293(humanembryo kidney 293 derived cell line)細(xì)胞。并觀察綠色熒光蛋白(green fluorescent protein,GFP)的表達(dá)。凍融法收集初代病毒液。取初代病毒液感染HEK293細(xì)胞,逐日觀察細(xì)胞病變(cytopathic effect,CPE),及有無彗星樣斑塊(FOCI)形成。并經(jīng)第三、四輪大量擴(kuò)增,收集第四輪病毒液,用腺病毒純化試劑盒進(jìn)行純化。 6.重組腺病毒滴度測(cè)定將293細(xì)胞培養(yǎng)在96孔板中細(xì)胞生長至70%-80%匯合時(shí),以不同稀釋倍數(shù)的各代病毒液感染細(xì)胞,感染48小時(shí)后,以GFP陽性細(xì)胞計(jì)數(shù)法測(cè)定病毒滴度。病毒滴度=熒光細(xì)胞數(shù)×病毒液的稀釋倍數(shù)/病毒液量。 結(jié)果:1.成功構(gòu)建了質(zhì)粒pGEM-T/C,限制性酶切鑒定及測(cè)序結(jié)果證明與預(yù)期結(jié)果相符合。 2.成功構(gòu)建了真核表達(dá)質(zhì)粒pcDNA3.0/C,酶切分析證明符合預(yù)期設(shè)計(jì)。 3.成功構(gòu)建重組穿梭質(zhì)粒pAdTrack-CMV/C,限制性酶切結(jié)果符合預(yù)期設(shè)計(jì)。 4.成功構(gòu)建重組腺病毒質(zhì)粒pAd/C,用PacI酶切得到約30Kb的大片段和一個(gè)4.5Kb的小片段,PCR鑒定目的基因長度與預(yù)期值相符。 5.經(jīng)293細(xì)胞轉(zhuǎn)染48小時(shí)后,熒光顯微鏡下觀察到報(bào)告基因GFP的表達(dá),重組腺病毒rAd/C包裝成功。初代病毒再次感染293細(xì)胞,在熒光顯微鏡下觀察到了逐日明顯變化的細(xì)胞病變和熒光聚集,且感染細(xì)胞后的各代病毒液均有彗星樣熒光斑塊形成。 6.重組腺病毒rAd/C大量擴(kuò)增后病毒滴度達(dá)到:6.8×108pfu/ml。 結(jié)論:本研究利用AdEasy腺病毒載體系統(tǒng)構(gòu)建并包裝重組腺病毒載體疫苗rAd/C,為丙型肝炎疫苗的研制奠定了基礎(chǔ)。
[Abstract]:Objective: hepatitis C is by hepatitis C virus (Hepatitis C, virus, HCV) caused by chronic liver disease, is a global health problem. At present, there is still no effective treatment. Therefore, the development of effective prevention and treatment of HCV vaccine, has become an urgent problem to be solved.
HCV belongs to the hepatitis C virus, the hepatitis C virus (hepatitis C, virus, HCV) high variability of different gene types and genotypes, especially the envelope gene variation is greater, so according to the traditional way of HCV vaccines is facing many difficulties. The exogenous gene encoding the target antigen by all a new type of vaccine recipients is expected to become a new way of HCV vaccine development. HCV core area (C area) is the most conservative and stable region, there have been reports that C antigen can induce high levels to hepatitis C virus specific antibody response and cytotoxic T lymphocyte (cytotoxic T, lymphocyte, CTL) activity of cells immunization is one of the main target antigen for HCV DNA vaccine.
Compared with the plasmid vector, replication defective adenovirus vector system (replication-deficient adenovirus vector system) has the ability to enter the host cell, wide host range, high transfection efficiency, gene expression and the characteristics of the high titer of virus showed advantage in vaccine carrier selection, become a promising gene transfer vector. In this study we use the recombinant replication defective adenovirus vector with HCV C antigen as target antigen effectively constructed expression of HCV core protein (HCV core protein) recombinant adenovirus rAd-C, to explore ways to develop new hepatitis C virus vaccine.
Methods: 1.. Amplification and identification of the target gene. The recombinant plasmid pEGFP-HCV/C was used as template to amplify the C target gene. The amplified product was connected with pGEM-T vector to construct plasmid pGEM-T/C. and to transform DH5 alpha E.coli. The plasmid was extracted, digested, identified and sequenced, and the recombinants were screened.
2. construction of eukaryotic expression plasmid pcDNA3.0/C pGEM-T/C plasmid with suitable restriction endonuclease digested gel fragment, and pcDNA3.0 plasmid by restriction endonuclease digestion of the same connection, through the transformation, screening and enzyme digestion, to construct eukaryotic expression plasmid pcDNA3.0/C.
3. construction of recombinant shuttle plasmid pGEM-T/C plasmid with suitable restriction endonuclease digested fragment recycling, the fragment with suitable restriction endonuclease digested Adenovirus Shuttle Vector pAdTrack-CMV. After connection transformation, screening and enzyme digestion, the recombinant shuttle plasmid pAdTrack-CMV/C.
4. recombinant adenovirus plasmid plasmid by restriction endonuclease PmeI linearized by AdEasy system, the two step of homologous recombination in Escherichia coli BJ-5183 with backbone plasmid pAdEasy-1, recombinant adenovirus plasmid pAd/C. by PacI enzyme digestion, recombinant plasmid was transformed into competent cells of E.coli DH5 alpha, a large number of alkaline lysis extraction method and using polyethylene glycol (polyethylene, glycol, PEG) was purified by precipitation method.
5. recombinant adenovirus packaging and amplification of recombinant adenovirus plasmid pAd/C by restriction endonuclease Pac-I linearized by cationic liposome transfection (HEK293 humanembryo kidney 293 derived cell line) cells. And observed the green fluorescent protein (green fluorescent, protein, GFP). The expression of freeze thawing method. The collected primary virus liquid primary virus liquid the infection of HEK293 cells, by observing the cytopathic effect (cytopathic effect, CPE), and there is no comet like (FOCI) and plaque formation. After third, fourth rounds of amplification, collecting fourth round virus liquid was purified with adenovirus purification kit.
6. the recombinant adenovirus titer of 293 cells were cultured in 96 well plate cell growth to 70%-80% confluence, with each generation of virus infected cells in liquid dilution, 48 h after infection, the virus titer was determined by counting the number of GFP positive cells. The virus titer = number of fluorescent cells * virus liquid dilution / virus liquid.
Results: 1. the plasmid pGEM-T/C was successfully constructed. Restriction endonuclease identification and sequencing results proved to be consistent with the expected results.
2. the eukaryotic expression plasmid pcDNA3.0/C was successfully constructed, and the enzyme cutting analysis proved to be in conformity with the expected design.
3. the recombinant shuttle plasmid pAdTrack-CMV/C was successfully constructed, and the restrictive enzyme cutting results were in conformity with the expected design.
4., the recombinant adenovirus plasmid pAd/C was successfully constructed, and a large fragment of 30Kb and a small fragment of 4.5Kb were obtained by PacI enzyme digestion. The length of the target gene was consistent with the expected value by PCR.
5. after 48 hours of 293 cells after transfection, fluorescence microscopy showed that expression of GFP reporter gene, recombinant adenovirus rAd/C packaged successfully. Primary virus re infection of 293 cells under the fluorescence microscope observed daily obvious change cytopathic and fluorescence aggregation, and the generation of virus infection have comet like fluorescent plaque cells after the formation.
The virus titer of 6. recombinant adenovirus rAd/C was reached: 6.8 x 108pfu/ml.
Conclusion: the recombinant adenovirus vector vaccine (rAd/C) was constructed and packaged by the AdEasy adenovirus vector system, which laid the foundation for the development of hepatitis C vaccine.

【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】：2010
【分類號(hào)】：R392.1

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