本文關(guān)鍵詞：可表達(dá)新型甲型H1N1流感病毒血凝素蛋白輔助病毒依賴型腺病毒載體的構(gòu)建及初步鑒定　出處：《北京交通大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 輔助病毒依賴型腺病毒載體 甲型H1N1流感病毒 血凝素蛋白 重組腺病毒 逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng) 免疫印跡

【摘要】：目的：新型甲型H1N1流感病毒是威脅人類健康的重要新發(fā)傳染病,雖然已有可用于該病毒預(yù)防和治療的疫苗及藥物,但現(xiàn)有的神經(jīng)氨酸酶抑制劑類抗病毒藥物和以傳統(tǒng)方法制備的流感病毒裂解疫苗均存在一定的局限性。近期研究表明,以重組腺病毒制備流感疫苗具有明顯的優(yōu)越性。本研究以全基因合成的方法獲得甲型H1N1流感病毒血凝素(Hemagglutinin, HA)基因,并以輔助病毒依賴型腺病毒(Helper-dependent adenoviral vector, HDAd)載體系統(tǒng)為基礎(chǔ),構(gòu)建可表達(dá)HA的重組HDAd,為進(jìn)一步的體內(nèi)免疫效果和免疫保護(hù)作用研究奠定基礎(chǔ)。 方法：根據(jù)GenBank上登錄的甲流H1N1流感病毒美國(guó)加利福尼亞毒株(A/California/07/2009(H1N1))基因序列,采用全基因合成的方法合成由1701個(gè)堿基組成的HA編碼基因。經(jīng)核酸序列分析證實(shí)后,將HA基因克隆至HDAd穿梭載體pSC11-CMV,經(jīng)限制性內(nèi)切酶分析后,進(jìn)一步將含有HA基因的表達(dá)盒克隆至HDAd骨架質(zhì)粒pSC15B,構(gòu)建pSC15B/HA重組質(zhì)粒,并以多組限制性內(nèi)切酶進(jìn)行分析鑒定。Pme I消化pSC15B/HA去除原核復(fù)制元件及抗性基因,獲得HDAd/HA DNA分子,經(jīng)磷酸鈣共沉淀法轉(zhuǎn)染293Cre4細(xì)胞,16h后感染輔助病毒,收獲HDAd/HA載體粗提液,隨后以HDAd/HA粗提液及輔助病毒連續(xù)共感染293Cre4細(xì)胞直至HDAd/HA達(dá)到復(fù)制極限。隨后,以HDAd/HA和輔助病毒感染293Cre4細(xì)胞大量制備HDAd/HA, CsCl密度梯度及等密度梯度兩次超速離心純化,利用透射電子顯微鏡觀察病毒形態(tài),分子生物學(xué)技術(shù)體外鑒定HDAd/HA表達(dá)情況。 結(jié)果：成功構(gòu)建了可表達(dá)甲流H1N1流感病毒HA蛋白的HDAd/HA載體,通過(guò)與輔助病毒共感染293Cre4細(xì)胞及CsCl梯度法超速離心制備了大量純化的HDAd/HA載體,透射電子顯微鏡觀察到腺病毒,體外感染293細(xì)胞,RT-PCR檢測(cè)到HA基因有轉(zhuǎn)錄 結(jié)論：應(yīng)用HDAd系統(tǒng),成功構(gòu)建了HDAd/HA重組腺病毒,并完成了該病毒的大量制備和純化,為體內(nèi)免疫學(xué)效力試驗(yàn)奠定了初步基礎(chǔ)。
[Abstract]:Objective: the new influenza A (H1N1) virus is an important emerging infectious disease threatening human health, although there are vaccines and drugs available for the prevention and treatment of the virus. However the existing neuraminidase inhibitors antiviral drugs and traditional methods of influenza virus lytic vaccine have some limitations. The preparation of influenza vaccine by recombinant adenovirus has obvious superiority. In this study, the hemagglutinin (HAN) gene of influenza A (H1N1) virus was obtained by the method of full gene synthesis. It is based on Helper-dependent adenoviral vector system of adjuvant virus-dependent adenovirus. The construction of recombinant HDAds expressing HA could lay a foundation for further study on immunological effects and immunoprotective effects in vivo. Methods: according to the gene sequence of A / A / 7 / 07 / 2009 H1 / N _ (1) of the H1N1 influenza virus A / A / 7 / 2009 which was registered on GenBank. The HA encoding gene was synthesized by whole gene synthesis. The HA gene was confirmed by nucleic acid sequence analysis and cloned into HDAd shuttle vector pSC11-CMV. After restriction endonuclease analysis, the expression cassette containing HA gene was further cloned into HDAd skeleton plasmid pSC15B to construct pSC15B/HA recombinant plasmid. The pSC15B/HA was digested by multiple restriction endonuclease to remove the prokaryotic replication elements and resistant genes, and the HDAd/HA DNA molecule was obtained. The 293Cre4 cells were transfected with calcium phosphate coprecipitation for 16 hours and then infected with covirus. The crude extract of HDAd/HA vector was harvested. Subsequently, 293Cre4 cells were co-infected with HDAd/HA extract and auxiliary virus until HDAd/HA reached the replication limit. HDAd- / HA, CsCl density gradient and isodensity gradient were purified from 293Cre4 cells infected with HDAd/HA and covirus respectively. The morphology of the virus was observed by transmission electron microscope (TEM) and the expression of HDAd/HA was identified by molecular biology in vitro. Results: the HDAd/HA vector expressing HA protein of H1N1 influenza virus was successfully constructed. A large number of purified HDAd/HA vectors were prepared by co-infection with auxiliary virus 293Cre4 and CsCl gradient centrifugation. The adenovirus was observed by transmission electron microscope. Transcription of HA gene detected by RT-PCR in 293 cells infected in vitro Conclusion: the recombinant adenovirus of HDAd/HA was successfully constructed by using HDAd system, and the preparation and purification of the recombinant adenovirus were completed, which laid a preliminary foundation for the in vivo immunological potency test.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373

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本文編號(hào)：1357663