本文選題：H5N1　切入點：密碼子優(yōu)化　出處：《中國疾病預(yù)防控制中心》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 自1997年H5N1禽流感病毒開始感染人以后,人間病例不斷發(fā)生,在泰國、印度尼西亞、中國和巴基斯坦還出現(xiàn)過有限的人傳人的病例,這表明H5N1流感病毒正在不斷進化,一旦H5N1流感病毒獲得人傳人的能力,引起的世界大流行將給人類帶來巨大的災(zāi)難。目前防控流感/禽流感最有效的措施仍為接種疫苗。而基于雞胚生產(chǎn)的季節(jié)性流感疫苗的生產(chǎn)能力是有限的,因此,如何縮小當(dāng)前疫苗生產(chǎn)能力與大流行期間疫苗巨大需求量的差距,提高疫苗產(chǎn)量是全球流感大流行疫苗研究的一個熱點問題。提高疫苗產(chǎn)量除了加強、擴大滅活疫苗的生產(chǎn)能力外,提高疫苗主要成分血凝素(HA)蛋白的表達量從而減少疫苗的使用量也是一種有效的方法。 本研究擬構(gòu)建針對H5N1流感病毒的DNA疫苗和腺病毒載體疫苗,通過優(yōu)化HA基因的密碼子,大大提高血凝素的表達水平,從而提高疫苗的免疫效果。另外,目前針對流感大流行疫苗的研究多采用Clade1的H5N1香港株或越南株,然而我國流行的H5N1流感病毒株基本上都是Clade2.3.4的毒株,與Clade1的毒株在抗原性上有很大不同,因此本研究選用中國的分離株具有重要意義。 本研究以我國人感染病例中分離到的、WHO推薦的Clade2.3.4疫苗候選株A/Anhui/1/2005(H5N1)為研究對象,首先按照人的偏愛密碼子將H5N1(A/Anhui/1/2005)流感病毒的HA基因進行優(yōu)化改造,以提高其表達量,經(jīng)全基因合成后,插入到真核表達載體pDC315中,構(gòu)建了真核表達質(zhì)粒pDC315-Mod.HA;將此質(zhì)粒和含野生HA基因的真核表達質(zhì)粒pDC315-Wt.HA分別轉(zhuǎn)染293T細(xì)胞,比較HA蛋白的表達量。在比較密碼子優(yōu)化效果之后,將優(yōu)化的HA基因Mod.HA插入DNA疫苗載體——pVR-1012,構(gòu)建了一種H5N1流感病毒的DNA疫苗pVR-Mod.HA;又利用AdMax~(TM)腺病毒包裝系統(tǒng),分別構(gòu)建了Ad-Wt.HA和Ad-Mod.HA兩個腺病毒載體疫苗;最后采用BALB/c小鼠單獨或者聯(lián)合免疫方法,評價不同疫苗和不同免疫策略所誘導(dǎo)的體液免疫及細(xì)胞免疫效果,并就疫苗誘導(dǎo)的中和抗體研究了其對8株不同H5N1流感病毒的交叉反應(yīng)性。 結(jié)果表明: 1)密碼子優(yōu)化后HA蛋白在293T細(xì)胞中的表達水平顯著提高,而且疫苗誘導(dǎo)小鼠產(chǎn)生的體液免疫和細(xì)胞免疫效果也有明顯提高; 2)本研究構(gòu)建的優(yōu)化的腺病毒載體疫苗不僅能夠有效誘導(dǎo)出中和抗體,而且對中國分離的不同年代不同地域的H5N1流感病毒普遍具有較強的中和作用; 3)單獨免疫兩次優(yōu)化的腺病毒載體疫苗能夠誘導(dǎo)較強的體液免疫和細(xì)胞免疫,而且都顯著優(yōu)于單獨免疫兩次DNA疫苗; 4) DNA疫苗初免-腺病毒載體疫苗加強的免疫策略是一種相對較好的聯(lián)合免疫策略,值得進一步研究; 5)從A/Anhui/1/2005(H5N1)HA全序列中篩選到了兩個能夠刺激BALB/c小鼠CD8+T淋巴細(xì)胞分泌IFN-γ的表位,即HA_(212-229):TYISVGTSTLNQRLVPKI和HA_(534-543):IYSTVASSLA,后者比前者強。
[Abstract]:Since 1997, when the H5N1 avian influenza virus began to infect people, human cases have continued to occur, and in Thailand, Indonesia, China and Pakistan, there have been limited cases of human transmission, indicating that the H5N1 influenza virus is evolving. Once the H5N1 influenza virus has acquired the ability to transmit human beings, At present, the most effective measures to prevent and control influenza / avian influenza are vaccination. The production capacity of seasonal influenza vaccine based on chicken embryo is limited, so the production capacity of seasonal influenza vaccine based on chicken embryo is limited. How to narrow the gap between the current vaccine production capacity and the huge demand for vaccines during the pandemic, and how to increase the vaccine production is a hot issue in global influenza pandemic vaccine research. In addition to expanding the production capacity of inactivated vaccines, it is also an effective method to increase the expression of hemagglutinin (HA) protein, which is the main component of the vaccine, so as to reduce the amount of vaccine used. In this study, DNA vaccine and adenovirus vector vaccine against H5N1 influenza virus were constructed. By optimizing the codon of HA gene, the level of hemagglutinin expression was greatly improved, and the immune effect of the vaccine was improved. At present, most of the influenza pandemic vaccine studies use Clade1 H5N1 strain of Hong Kong or Vietnam strain. However, the H5N1 influenza virus strains in China are basically Clade2.3.4 strains, which are very different from Clade1 strains in antigenicity. Therefore, it is of great significance to select Chinese isolates in this study. In this study, the candidate strain of Clade2.3.4 vaccine, A / Anhui / 1 / 2005, recommended by WHO, which was isolated from human infection cases in China, was used as the research object. Firstly, the HA gene of H5N1 N1 / Anhui / 1 / 2005) influenza virus was optimized and modified according to the human preferred codon to increase its expression. After the whole gene was synthesized and inserted into the eukaryotic expression vector pDC315, the eukaryotic expression plasmid pDC315-Mod.HAwas constructed, and the eukaryotic expression plasmid pDC315-Wt.HA containing wild HA gene was transfected into 293T cells, respectively. After comparing the effect of codon optimization, the optimized HA gene Mod.HA was inserted into the DNA vaccine vector pVR-1012 to construct a H5N1 influenza virus DNA vaccine pVR-Mod.HA. Two adenovirus vector vaccines, Ad-Wt.HA and Ad-Mod.HA, were constructed, and the humoral and cellular immunity induced by different vaccines and different immune strategies were evaluated by BALB/c mice alone or in combination. The cross-reactivity of neutralizing antibody to 8 different H5N1 influenza viruses was studied. The results show that:. 1) the expression of HA protein in 293T cells was significantly increased after codon optimization, and the humoral and cellular immunity of mice induced by vaccine was also improved. 2) the optimized adenovirus vector vaccine can not only effectively induce neutralizing antibody, but also have strong neutralizing effect on H5N1 influenza virus isolated in different years and different regions in China. 3) Adenovirus-vector vaccine could induce strong humoral and cellular immunity, and it was significantly better than DNA vaccine twice immunized alone. 4) the immunization strategy of the primary immune-adenovirus vector vaccine reinforced by DNA vaccine is a relatively good strategy of combined immunization, which deserves further study. 5) two epitopes, HA_(212-229):TYISVGTSTLNQRLVPKI and HASP 54-543: IYSTVASSLAs, which can stimulate the secretion of IFN- 緯 by CD8 T lymphocytes in BALB/c mice, were screened from the A / Anhui / 1 / 2005 H _ 5H _ 5N _ 1H _ 1 HA sequence, the latter being stronger than the former.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392.1

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本文編號：1556885