【摘要】：2013年于我國長(zhǎng)三角地區(qū)首次分離的H7N9新型禽流感病毒(Avian influenza virus,AIV),歷經(jīng)4年的不斷進(jìn)化,已從最初的對(duì)家禽無或低致病性衍化出了部分高致病性的毒株。這些變異株在HA蛋白裂解位點(diǎn)含有四個(gè)連續(xù)堿性氨基酸(PKRKRTAR/GLF)的插入,符合高致病性禽流感(Highly pathogenic avian influenza，HPAI)病毒的分子特征,嚴(yán)重危害養(yǎng)殖業(yè)的健康發(fā)展。此外,H7N9亞型AIV還可以直接感染人。根據(jù)世界衛(wèi)生組織最新的統(tǒng)計(jì)數(shù)據(jù),截至2017年5月31日,經(jīng)實(shí)驗(yàn)室確診的人感染H7N9病例已達(dá)1532多例,其中死亡581人。盡管目前仍缺乏足夠證據(jù)表明H7N9病毒能夠在人與人之間進(jìn)行有效傳播,但其造成流感大流行的風(fēng)險(xiǎn)依然很高,對(duì)公共衛(wèi)生安全的危害不容小覷。在我國,疫苗接種仍是現(xiàn)階段防控HPAI的基本措施之一,然而目前臨床尚無針對(duì)家禽的H7N9疫苗,因此亟需研制相應(yīng)的疫苗從源頭上防控疫情,進(jìn)而減少由H7N9亞型AIV引發(fā)的對(duì)畜禽生產(chǎn)和公眾健康的危害。傳統(tǒng)的滅活疫苗主要激發(fā)體液免疫,缺乏細(xì)胞免疫應(yīng)答,其免疫效果往往會(huì)由于母源抗體水平高、疫苗株與流行株的抗原匹配性差等因素而受到限制。相比而言,新型的載體活疫苗則具有諸多優(yōu)勢(shì),可以克服傳統(tǒng)滅活疫苗的一些弊端。例如,以火雞皰疹病毒(HVT)為載體表達(dá)保護(hù)性抗原的重組活疫苗具有能同時(shí)誘導(dǎo)細(xì)胞和體液免疫、保護(hù)周期長(zhǎng)以及降低感染后排毒等特點(diǎn),是一種較為理想的禽用新型載體疫苗。然而,前期有相關(guān)研究指出,將AIV(H7N1亞型)的主要保護(hù)性抗原HA插入HVT所制備的重組病毒rHVT-H7HA,僅能使約73%的SPF雞獲得死亡保護(hù)。另有多項(xiàng)研究表明,啟動(dòng)子的選擇和HA蛋白本身的免疫原性可能是影響HVT重組病毒免疫效果的重要因素。因此,本研究通過選擇不同啟動(dòng)子和增強(qiáng)HA抗原性的策略,構(gòu)建了一系列中間轉(zhuǎn)移質(zhì)粒;進(jìn)而通過同源重組和嘗試CRISPR/Cas9介導(dǎo)的靶向基因編輯技術(shù),構(gòu)建了表達(dá)H7N9亞型AIV HA蛋白的重組HVT疫苗,并對(duì)其在SPF雞上的免疫效力進(jìn)行了測(cè)定。本研究首先克隆了 HVT內(nèi)源性的gB啟動(dòng)子(HgB),同時(shí)選擇了外源性的CMV強(qiáng)啟動(dòng)子進(jìn)行對(duì)比研究。隨后,基于1株低致病性H7N9亞型AIV的HA基因,為提高其氨基酸在雞體內(nèi)的翻譯效率,進(jìn)行了雞源(Gallus gallus)密碼子的優(yōu)化(OHA),構(gòu)建了含有不同啟動(dòng)子的中間轉(zhuǎn)移質(zhì)粒pHOH(HgB-OHA)和pVOH(CMV-OHA)。進(jìn)一步于OHA蛋白的N端添加MHCI類分子的信號(hào)肽(MHCIss)并替換C端的跨膜-胞內(nèi)區(qū)(MITD)為MHC I類分子的對(duì)應(yīng)區(qū)域,以增強(qiáng)MHCI類抗原肽的遞呈效率,優(yōu)化OHA為OHAM;并繼續(xù)在OHAM的C端添加WPRE(土撥鼠肝炎轉(zhuǎn)錄后調(diào)控元件)序列,以提高蛋白質(zhì)翻譯的速率,構(gòu)建了嵌合增強(qiáng)型的中間轉(zhuǎn)移質(zhì)粒pHMW(HgB-OHAM-WPRE)和pVMW(CMV-OHAM-WPRE)。使用商品化的針對(duì)H7N9病毒HA的單克隆抗體,經(jīng)間接免疫熒光試驗(yàn)(IFA)和蛋白質(zhì)免疫印跡(WB)鑒定,上述4個(gè)中間轉(zhuǎn)移質(zhì)粒均能夠成功表達(dá)70kDa左右的HA蛋白。為了進(jìn)一步獲得表達(dá)HA蛋白的重組HVT,將上述構(gòu)建的中間轉(zhuǎn)移質(zhì)粒與實(shí)驗(yàn)室前期構(gòu)建好的表達(dá)GFP蛋白的重組HVT(rHVT-GFP，，GFP插入至HVT復(fù)制非必需區(qū)的US2區(qū))基因組DNA,使用磷酸鈣法共轉(zhuǎn)染CEF細(xì)胞進(jìn)行同源重組,經(jīng)篩選、純化、鑒定后,獲得了兩株含有HgB啟動(dòng)子的重組HVT:rHOH和rHMW;同時(shí),為了提高同源重組的效率,嘗試?yán)肅RISPR/Cas9技術(shù)獲得了一株含有CMV啟動(dòng)子的重組HVT:rVMW。經(jīng)IFA、WB和測(cè)序鑒定,構(gòu)建的3株重組HVT均能夠成功表達(dá)外源HA蛋白。接下來,在CEF細(xì)胞上測(cè)定了 rHOH和rHMW的遺傳穩(wěn)定性并利用激光共聚焦顯微鏡觀察了這兩株重組HVT表達(dá)的HA蛋白在感染細(xì)胞中的定位,結(jié)果顯示,rHOH和rHMW歷經(jīng)連續(xù)20代的細(xì)胞傳代后仍能穩(wěn)定表達(dá)HA蛋白,生長(zhǎng)速度與野生型HVT無明顯差異;其中rHOH表達(dá)的HA主要定位于細(xì)胞漿內(nèi),而rHMW表達(dá)的HA同時(shí)定位于細(xì)胞漿和細(xì)胞膜表面。進(jìn)一步將構(gòu)建的rHOH和rHMW以不同劑量分別免疫1日齡SPF雞后進(jìn)行攻毒保護(hù)試驗(yàn),評(píng)價(jià)了重組HVT的免疫效力。結(jié)果顯示,免疫后6周,rHMW各免疫組的血清抗體陽轉(zhuǎn)率在62.5%-72%之間,而rHOH各免疫組的血清陽轉(zhuǎn)率≤62.5%;選取實(shí)驗(yàn)室2017年分離鑒定的一株HPAIH7N9病毒以105TCIDs0進(jìn)行攻毒,rHMW免疫組可提供約77.8%的臨床保護(hù)。綜上所述,本研究成功構(gòu)建了能夠表達(dá)H7N9亞型禽流感病毒HA蛋白的重組火雞皰疹病毒,并對(duì)其在SPF雞上的免疫效力進(jìn)行了評(píng)價(jià)。盡管rHOH和rHMW重組病毒的臨床保護(hù)效果有待進(jìn)一步提高,但本研究通過選擇利用不同的啟動(dòng)子、對(duì)表達(dá)的外源蛋白進(jìn)行密碼子優(yōu)化和添加促進(jìn)蛋白翻譯的作用元件等新型疫苗研制策略,以及建立的CRISPR/Cas9技術(shù)平臺(tái),都將為基于HVT以及其他禽皰疹病毒載體疫苗的相關(guān)研究奠定基礎(chǔ)。
[Abstract]:The first isolated H7N9 avian influenza virus (AIV), which was first isolated in the Yangtze River Delta area in China in 2013, has evolved over four years, and has developed some highly pathogenic strains from the original or low pathogenicity of the poultry. These variants have four continuous basic amino acids (PKRKRTAR/ GLF) inserted in the HA protein cleavage site, which are in accordance with the molecular characteristics of highly pathogenic avian influenza (HPAI) virus and seriously endanger the healthy development of the breeding industry. In addition, the H7N9 subtype AIV can also be directly infected. According to the latest statistics from the World Health Organization, as of 31 May 2017, the number of human-infected H7N9 cases confirmed by the laboratory reached 1532, including 581 deaths. While there is still a lack of sufficient evidence to suggest that the H7N9 virus is able to spread effectively between human and human beings, the risk of the pandemic is still high, and the harm to public health security cannot be underestimated. In our country, the vaccination is still one of the basic measures to prevent and control the HPAI. However, there is no H7N9 vaccine for poultry at present. Therefore, it is urgent to develop the corresponding vaccine to prevent and control the disease from the source, so as to reduce the harm to the production of the livestock and poultry and the public health caused by the H7N9 subtype AIV. The traditional inactivated vaccine mainly stimulates the humoral immunity and lacks the cellular immune response, and the immune effect of the inactivated vaccine is often limited due to the high level of the maternal antibody, poor matching of the vaccine strain and the antigen of the epidemic strain, and the like. In contrast, the novel vector live vaccine has many advantages, and can overcome some disadvantages of the traditional inactivated vaccine. For example, a recombinant live vaccine expressing a protective antigen by using a turkey herpesvirus (HVT) as a carrier has the characteristics of simultaneously inducing cell and body fluid immunity, long protection period, and reducing toxin and the like after infection, and is an ideal novel carrier vaccine for poultry. However, the previous studies indicated that the main protective antigen HA of the AIV (H7N1 subtype) was inserted into the recombinant virus rHVT-H7HA prepared by HVT, and only about 73% of the SPF chickens were protected from death. Several other studies have shown that the selection of the promoter and the immunogenicity of the HA protein may be an important factor in the immune response of the HVT recombinant virus. Therefore, a series of intermediate transfer plasmids were constructed by selecting different promoters and strategies to enhance the antigenicity of HA, and then a recombinant HVT vaccine expressing the AIV HA protein of the H7N9 subtype was constructed by homologous recombination and a targeted gene editing technique mediated by CRISPR/ Cas9. And the immune efficacy on the SPF chicken is determined. In this study, the endogenous gB promoter (HgB) of HVT was cloned, and the exogenous CMV strong promoter was selected for comparative study. Subsequently, based on the HA gene of 1 strain of low-pathogenicity H7N9 subtype AIV, in order to improve the translation efficiency of the amino acid in the chicken, the optimization of the codon of the chicken feed was carried out (OHA), and the intermediate transfer plasmid pHCO (HgB-OHA) and pVOH (CMV-OHA) containing different promoters were constructed. the signal peptide (MHCIss) of the MHCI type molecule is further added at the N end of the OHA protein and the transmembrane-intracellular region (MITD) at the C end is replaced by the corresponding region of the MHC class I molecule so as to enhance the delivery efficiency of the MHC class I molecule, and the OSHA is optimized to be OHAM; In order to improve the rate of protein translation, a chimeric enhanced intermediate transfer plasmid pHMW (HgB-OHM-WPRE) and pVMW (CMV-OHAM-WPRE) were constructed. A commercial monoclonal antibody against H7N9 virus HA was used to identify the HA protein of about 70 kDa by indirect immunofluorescence assay (IFA) and protein immunoblotting (WB). In order to further obtain the recombinant HVT expressing the HA protein, the constructed intermediate transfer plasmid and the recombinant HVT (rHVT-GFP, GFP into the US2 region of the HVT replication non-required region) constructed in the early stage of the laboratory were inserted into the genomic DNA, and the CEF cells were co-transfected with the calcium phosphate method for homologous recombination, In order to improve the efficiency of homologous recombination, a recombinant HVT: rVMW containing the CMV promoter was obtained by using the CRISPR/ Cas9 technique. 3 recombinant HVT constructed by IFA, WB and sequencing can successfully express the foreign HA protein. Next, the genetic stability of rHOH and rHHMW was determined on the CEF cells and the localization of the two recombinant HVT-expressing HA proteins in the infected cells was observed by using a laser confocal microscope. The results showed that the rHOH and rHMW were able to stably express the HA protein after the cell passage of 20 generations. There was no significant difference between the growth rate and the wild type HVT; the HA expressed in rHOH was mainly located in the cytoplasm, while the rHMW-expressed HA was located at both the cell and cell membrane surfaces. The rHOH and rHHMW were respectively immunized with different doses of SPF chickens at 1 day of age, and the immune efficacy of the recombinant HVT was evaluated. The results showed that the positive rate of serum antibody of rHOH group was between 62.5% and 72%, while the seropositive rate of rHOH group was 62.5%, and a HPAIH7N9 virus isolated and identified by the laboratory in 2017 was challenged with 105 TCIDs0, and the rHMW immune group could provide about 77.8% of clinical protection. To sum up, the present study successfully constructed a recombinant human herpesvirus capable of expressing the HA protein of the H7N9 subtype avian influenza virus, and evaluated the immune efficacy of the H7N9 subtype avian influenza virus HA protein. Although the clinical protective effect of rHOH and rHMW recombinant virus is to be further improved, the present study adopts a novel vaccine development strategy such as codon optimization of the expressed foreign protein and the addition of a functional element for promoting protein translation by selecting different promoters, And the established CRISPR/ Cas9 technical platform will lay the foundation for relevant research based on HVT and other avian herpesvirus vector vaccines.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李呈軍;陳化蘭;;反向遺傳學(xué)技術(shù)在流感病毒研究和防控中的應(yīng)用[J];中國科學(xué):生命科學(xué);2015年10期

2 顧敏;彭大新;劉秀梵;;我國H9N2亞型禽流感病毒的流行和進(jìn)化特點(diǎn)[J];生命科學(xué);2015年05期

3 陳曦;于桂梅;劉景利;王金良;姜永萍;葛金英;步志高;;表達(dá)H5N1亞型禽流感病毒HA蛋白重組新城疫病毒的構(gòu)建以及密碼子優(yōu)化的HA免疫增強(qiáng)作用[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2014年08期

4 高冬妮;平文祥;金麗穎;沈萬力;宋剛;唐曉艷;安琦;申燕;葛菁萍;;以具有WPRE調(diào)控元件的桿狀病毒為載體在雞胚原代細(xì)胞中表達(dá)新城疫病毒F基因[J];微生物學(xué)報(bào);2014年04期

5 劉正偉;李慧敏;黃妙容;郭凱;王建麗;李延鵬;陳瑞愛;;不同啟動(dòng)子表達(dá)載體的構(gòu)建及其體外活性分析[J];中國畜牧獸醫(yī);2014年02期

6 呂玲;陳連頤;;積跬步行千里 創(chuàng)新疫苗顯威力——梅里亞威力克上市發(fā)布會(huì)在浙江杭州舉行[J];中國家禽;2011年09期

7 蘭德松;石星明;王云峰;劉長(zhǎng)軍;王玫;崔紅玉;田國彬;李繼松;童光志;;利用Rde/ET技術(shù)構(gòu)建表達(dá)H5亞型禽流感病毒HA基因的重組火雞皰疹病毒[J];微生物學(xué)報(bào);2009年01期

8 邱亞峰;葛菲菲;徐學(xué)清;陳溥言;;馬立克氏病病毒CVI988株病毒囊膜糖蛋白B啟動(dòng)子的克隆及活性分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2006年05期

9 劉紅梅;秦愛建;劉岳龍;金文杰;葉建強(qiáng);陳鴻軍;邵紅霞;李迎曉;;表達(dá)IBDV VP2融合蛋白的重組MDV的構(gòu)建及其免疫特性[J];生物工程學(xué)報(bào);2006年03期

10 邱亞峰;葛菲菲;徐學(xué)清;陳溥言;;不同異源啟動(dòng)子與MDVgB啟動(dòng)子及其復(fù)合啟動(dòng)子的活性比較[J];微生物學(xué)報(bào);2006年02期

相關(guān)博士學(xué)位論文 前1條

1 張小榮;2009-2011年國內(nèi)傳染性支氣管炎病毒分子流行病學(xué)研究及以馬立克病毒為載體的基因工程疫苗研制[D];揚(yáng)州大學(xué);2012年

相關(guān)碩士學(xué)位論文 前1條

1 梁苑燕;豬偽狂犬病病毒gE/TK基因缺失株的構(gòu)建及免疫效力的研究[D];揚(yáng)州大學(xué);2012年

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