表达H7N9亚型禽流感病毒HA蛋白重组火鸡疱疹病毒的构建与免疫效力测定

发布时间：2019-03-17 10:58

【摘要】：2013年于我国长三角地区首次分离的H7N9新型禽流感病毒(Avian influenza virus,AIV),历经4年的不断进化,已从最初的对家禽无或低致病性衍化出了部分高致病性的毒株。这些变异株在HA蛋白裂解位点含有四个连续碱性氨基酸(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,严重危害养殖业的健康发展。此外,H7N9亚型AIV还可以直接感染人。根据世界卫生组织最新的统计数据,截至2017年5月31日,经实验室确诊的人感染H7N9病例已达1532多例,其中死亡581人。尽管目前仍缺乏足够证据表明H7N9病毒能够在人与人之间进行有效传播,但其造成流感大流行的风险依然很高,对公共卫生安全的危害不容小觑。在我国,疫苗接种仍是现阶段防控HPAI的基本措施之一,然而目前临床尚无针对家禽的H7N9疫苗,因此亟需研制相应的疫苗从源头上防控疫情,进而减少由H7N9亚型AIV引发的对畜禽生产和公众健康的危害。传统的灭活疫苗主要激发体液免疫,缺乏细胞免疫应答,其免疫效果往往会由于母源抗体水平高、疫苗株与流行株的抗原匹配性差等因素而受到限制。相比而言,新型的载体活疫苗则具有诸多优势,可以克服传统灭活疫苗的一些弊端。例如,以火鸡疱疹病毒(HVT)为载体表达保护性抗原的重组活疫苗具有能同时诱导细胞和体液免疫、保护周期长以及降低感染后排毒等特点,是一种较为理想的禽用新型载体疫苗。然而,前期有相关研究指出,将AIV(H7N1亚型)的主要保护性抗原HA插入HVT所制备的重组病毒rHVT-H7HA,仅能使约73%的SPF鸡获得死亡保护。另有多项研究表明,启动子的选择和HA蛋白本身的免疫原性可能是影响HVT重组病毒免疫效果的重要因素。因此,本研究通过选择不同启动子和增强HA抗原性的策略,构建了一系列中间转移质粒;进而通过同源重组和尝试CRISPR/Cas9介导的靶向基因编辑技术,构建了表达H7N9亚型AIV HA蛋白的重组HVT疫苗,并对其在SPF鸡上的免疫效力进行了测定。本研究首先克隆了 HVT内源性的gB启动子(HgB),同时选择了外源性的CMV强启动子进行对比研究。随后,基于1株低致病性H7N9亚型AIV的HA基因,为提高其氨基酸在鸡体内的翻译效率,进行了鸡源(Gallus gallus)密码子的优化(OHA),构建了含有不同启动子的中间转移质粒pHOH(HgB-OHA)和pVOH(CMV-OHA)。进一步于OHA蛋白的N端添加MHCI类分子的信号肽(MHCIss)并替换C端的跨膜-胞内区(MITD)为MHC I类分子的对应区域,以增强MHCI类抗原肽的递呈效率,优化OHA为OHAM;并继续在OHAM的C端添加WPRE(土拨鼠肝炎转录后调控元件)序列,以提高蛋白质翻译的速率,构建了嵌合增强型的中间转移质粒pHMW(HgB-OHAM-WPRE)和pVMW(CMV-OHAM-WPRE)。使用商品化的针对H7N9病毒HA的单克隆抗体,经间接免疫荧光试验(IFA)和蛋白质免疫印迹(WB)鉴定,上述4个中间转移质粒均能够成功表达70kDa左右的HA蛋白。为了进一步获得表达HA蛋白的重组HVT,将上述构建的中间转移质粒与实验室前期构建好的表达GFP蛋白的重组HVT(rHVT-GFP，，GFP插入至HVT复制非必需区的US2区)基因组DNA,使用磷酸钙法共转染CEF细胞进行同源重组,经筛选、纯化、鉴定后,获得了两株含有HgB启动子的重组HVT:rHOH和rHMW;同时,为了提高同源重组的效率,尝试利用CRISPR/Cas9技术获得了一株含有CMV启动子的重组HVT:rVMW。经IFA、WB和测序鉴定,构建的3株重组HVT均能够成功表达外源HA蛋白。接下来,在CEF细胞上测定了 rHOH和rHMW的遗传稳定性并利用激光共聚焦显微镜观察了这两株重组HVT表达的HA蛋白在感染细胞中的定位,结果显示,rHOH和rHMW历经连续20代的细胞传代后仍能稳定表达HA蛋白,生长速度与野生型HVT无明显差异;其中rHOH表达的HA主要定位于细胞浆内,而rHMW表达的HA同时定位于细胞浆和细胞膜表面。进一步将构建的rHOH和rHMW以不同剂量分别免疫1日龄SPF鸡后进行攻毒保护试验,评价了重组HVT的免疫效力。结果显示,免疫后6周,rHMW各免疫组的血清抗体阳转率在62.5%-72%之间,而rHOH各免疫组的血清阳转率≤62.5%;选取实验室2017年分离鉴定的一株HPAIH7N9病毒以105TCIDs0进行攻毒,rHMW免疫组可提供约77.8%的临床保护。综上所述,本研究成功构建了能够表达H7N9亚型禽流感病毒HA蛋白的重组火鸡疱疹病毒,并对其在SPF鸡上的免疫效力进行了评价。尽管rHOH和rHMW重组病毒的临床保护效果有待进一步提高,但本研究通过选择利用不同的启动子、对表达的外源蛋白进行密码子优化和添加促进蛋白翻译的作用元件等新型疫苗研制策略,以及建立的CRISPR/Cas9技术平台,都将为基于HVT以及其他禽疱疹病毒载体疫苗的相关研究奠定基础。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

【参考文献】