以HBC和HPV16L1為載體的流感通用疫苗研究
發(fā)布時(shí)間：2018-11-05 12:25
【摘要】： 背景知識(shí): 流感(Influenza)是由流行性感冒病毒(Influenza virus)感染引起的急性呼吸道傳染病。流感病毒屬于正粘病毒科,可感染人和多種動(dòng)物。流感病毒抗原性易發(fā)生變異,抗原的漂移和轉(zhuǎn)換給流感的防治帶來(lái)了較大困難。接種疫苗仍然是預(yù)防和控制流感的主要手段。目前使用的疫苗包括全病毒疫苗、裂解疫苗和減毒活疫苗,但都需要每年更換疫苗株接種。因此,發(fā)展流感通用疫苗,實(shí)現(xiàn)幾年接種免疫一次是流感疫苗研究發(fā)展的方向。 目的: 本實(shí)驗(yàn)構(gòu)建以乙肝核心蛋白(HBC)和人乳頭瘤病毒主要衣殼蛋白(HPV16 L1)為載體的甲型流感病毒M2基因胞外區(qū)(M2e)通用疫苗桿粒,利用昆蟲(chóng)細(xì)胞桿狀病毒表達(dá)系統(tǒng),進(jìn)行初步的蛋白表達(dá),為發(fā)展安全、有效、穩(wěn)定的流感通用疫苗奠定基礎(chǔ)。 方法與結(jié)果: 1.將甲型流感病毒M2e基因序列設(shè)計(jì)在引物上,利用PCR技術(shù)分別與HBC和HPV16 L1相連。將M2e-HBC和M2e-HPV16 L1序列分別克隆到供體質(zhì)粒pFastBac HTA上,構(gòu)建兩個(gè)重組質(zhì)粒,分別為M2e-HBC-pFastBac HTA和M2e-HPV16 L1-pFastBac HTA。 2.用E.coli DH10Bac中的穿梭載體Bacmid與重組質(zhì)粒轉(zhuǎn)座,使含有M2e-HBC和M2e-HPV16 L1基因的表達(dá)盒插入Bacmid的attTn7的接受位點(diǎn),獲得重組Bacmid。 3.將挑選的陽(yáng)性重組子DNA轉(zhuǎn)染sf9細(xì)胞,取上清和細(xì)胞,上清繼續(xù)擴(kuò)增病毒,用空斑試驗(yàn)測(cè)定病毒效價(jià);免疫熒光法檢測(cè)蛋白表達(dá);細(xì)胞做破胞處理后,將上清和裂解液進(jìn)行SDS-PAGE分析;將感染病毒的細(xì)胞裂解后透射電鏡觀察。結(jié)果證明,M2e-HBC和M2e-HPV16 L1基因有蛋白表達(dá),而對(duì)照呈現(xiàn)陰性;電鏡可見(jiàn)細(xì)胞內(nèi)病毒樣顆粒(VLP)形成。 結(jié)論: 構(gòu)建了含有M2e-HBC和M2e-HPV16 L1的桿粒。利用Bac-to-Bac桿狀病毒表達(dá)系統(tǒng),將構(gòu)建好的桿粒轉(zhuǎn)染sf9昆蟲(chóng)細(xì)胞,得到含有重組桿狀病毒的上清,擴(kuò)增病毒,感染細(xì)胞后,表達(dá)出M2e-HBC和M2e-HPV16 L1目的蛋白。流感病毒樣顆�？乖璏2e-HBC和M2e-HPV16 L1高效表達(dá),為流感病毒通用疫苗的研制奠定了基礎(chǔ)。
[Abstract]:Background: influenza (Influenza) is an acute respiratory infection caused by influenza virus (Influenza virus) infection. Influenza viruses belong to the orthomyxovirus family and infect humans and many animals. The antigenicity of influenza viruses is easy to mutate, and the drift and transformation of antigens bring great difficulties to the prevention and treatment of influenza. Vaccination remains the primary means of influenza prevention and control. Vaccines currently in use include whole-virus vaccines, lytic vaccines and live attenuated vaccines, but require annual replacement of the vaccine strain. Therefore, it is the direction of influenza vaccine research and development to develop a universal influenza vaccine and to vaccinate once in a few years. Objective: to construct a universal vaccine of influenza A virus M2 gene extracellular region (M2e) using hepatitis B core protein (HBC) and human papillomavirus main capsid protein (HPV16 L1) as vectors. The baculovirus expression system of insect cells was used to carry out the preliminary protein expression, which laid the foundation for the development of a safe, effective and stable universal influenza vaccine. Methods and results: 1. The M2e gene sequence of influenza A virus was designed on primer and linked to HBC and HPV16 L1 by PCR technique. M2e-HBC and M2e-HPV16 L1 sequences were cloned into donor plasmid pFastBac HTA, and two recombinant plasmids, M2e-HBC-pFastBac HTA and M2e-HPV16 L1-pFastBac HTA., were constructed. 2. By transposing the shuttle vector Bacmid in E.coli DH10Bac with the recombinant plasmid, the expression box containing M2e-HBC and M2e-HPV16 L1 gene was inserted into the acceptance site of Bacmid attTn7, and the recombinant Bacmid. was obtained. 3. The positive recombinant DNA was transfected into sf9 cells. The supernatant and the supernatant were used to amplify the virus, the titer of the virus was determined by plaque test, and the protein expression was detected by immunofluorescence assay. The supernatant and the lytic fluid were analyzed by SDS-PAGE, and the infected cells were observed by transmission electron microscope. The results showed that M2e-HBC and M2e-HPV16 L1 gene had protein expression, but the control showed negative expression, and the virus like (VLP) was found in the cells under electron microscope. Conclusion: rods containing M2e-HBC and M2e-HPV16 L1 were constructed. The recombinant baculovirus supernatant was obtained by transfection of sf9 insect cells with Bac-to-Bac baculovirus expression system. The recombinant baculovirus supernatant was amplified and the target proteins of M2e-HBC and M2e-HPV16 L1 were expressed after infection. The high expression of influenza virus-like particle antigen M2e-HBC and M2e-HPV16 L1 lays a foundation for the development of influenza virus vaccine.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392
本文編號(hào)：2312076
[Abstract]:Background: influenza (Influenza) is an acute respiratory infection caused by influenza virus (Influenza virus) infection. Influenza viruses belong to the orthomyxovirus family and infect humans and many animals. The antigenicity of influenza viruses is easy to mutate, and the drift and transformation of antigens bring great difficulties to the prevention and treatment of influenza. Vaccination remains the primary means of influenza prevention and control. Vaccines currently in use include whole-virus vaccines, lytic vaccines and live attenuated vaccines, but require annual replacement of the vaccine strain. Therefore, it is the direction of influenza vaccine research and development to develop a universal influenza vaccine and to vaccinate once in a few years. Objective: to construct a universal vaccine of influenza A virus M2 gene extracellular region (M2e) using hepatitis B core protein (HBC) and human papillomavirus main capsid protein (HPV16 L1) as vectors. The baculovirus expression system of insect cells was used to carry out the preliminary protein expression, which laid the foundation for the development of a safe, effective and stable universal influenza vaccine. Methods and results: 1. The M2e gene sequence of influenza A virus was designed on primer and linked to HBC and HPV16 L1 by PCR technique. M2e-HBC and M2e-HPV16 L1 sequences were cloned into donor plasmid pFastBac HTA, and two recombinant plasmids, M2e-HBC-pFastBac HTA and M2e-HPV16 L1-pFastBac HTA., were constructed. 2. By transposing the shuttle vector Bacmid in E.coli DH10Bac with the recombinant plasmid, the expression box containing M2e-HBC and M2e-HPV16 L1 gene was inserted into the acceptance site of Bacmid attTn7, and the recombinant Bacmid. was obtained. 3. The positive recombinant DNA was transfected into sf9 cells. The supernatant and the supernatant were used to amplify the virus, the titer of the virus was determined by plaque test, and the protein expression was detected by immunofluorescence assay. The supernatant and the lytic fluid were analyzed by SDS-PAGE, and the infected cells were observed by transmission electron microscope. The results showed that M2e-HBC and M2e-HPV16 L1 gene had protein expression, but the control showed negative expression, and the virus like (VLP) was found in the cells under electron microscope. Conclusion: rods containing M2e-HBC and M2e-HPV16 L1 were constructed. The recombinant baculovirus supernatant was obtained by transfection of sf9 insect cells with Bac-to-Bac baculovirus expression system. The recombinant baculovirus supernatant was amplified and the target proteins of M2e-HBC and M2e-HPV16 L1 were expressed after infection. The high expression of influenza virus-like particle antigen M2e-HBC and M2e-HPV16 L1 lays a foundation for the development of influenza virus vaccine.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 朱建國(guó);A型流感病毒基質(zhì)蛋白研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2004年02期
，本文編號(hào)：2312076
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