【作者】 李凯；

【导师】 陈振海； 金宁一；

【作者基本信息】 扬州大学 ， 预防兽医学， 2021， 硕士

【摘要】 流行性感冒简称流感,是由流感病毒(Ⅳ)引起的一种急性呼吸系统疾病[1]。流感爆发主要是由A型流感病毒亚型引起的,部分亚型已在人群中传播。而流感大流行是由人-禽流感病毒重组病毒引起,其中A型H1N1、H3N2和B型流感病毒感染人偏多,每年呈季节性流行,常伴随咳嗽、高烧、流鼻涕、食欲减退以及全身肌肉酸痛等症状。目前,禽流感病毒H5N1和H7N9已突破种间屏障,具备人畜共患特征,具有引发大流行的可能[2]。流感病毒易发生抗原漂移或抗原转变,导致流感病毒多亚型共存,不同亚型间交叉保护性不好。目前的流感疫苗不能对抗原变异的流感毒株提供有效的保护,故研制一种可以同时预防多种亚型的流感病毒的疫苗迫在眉睫。本研究主要利用铁蛋白纳米粒子自组装和可修饰的特性,将流感病毒保守抗原HA2和M2e插入到铁蛋白纳米粒子的N端,通过原核表达系统和昆虫-杆状病毒表达系统表达基于铁蛋白的多表位流感纳米粒。佐剂 Alum、MF59、CpG、Poly:IC、MF59+CpG、MF59+Poly:IC 分别与多表位流感纳米粒混合后,制成疫苗并免疫小鼠。通过免疫的效果,筛选出适用于多表位流感纳米疫苗的佐剂。通过攻毒实验评价了各组疫苗的保护效果,为通用型流感纳米疫苗成功研发奠定基础。1.基于铁蛋白的多表位流感纳米疫苗的原核表达及鉴定将H1/H3/B亚型流感病毒HA2和M2e保守抗原基因串联Ferritin基因,形成HA2-M2e-Ferritin 基因(简称 HM2e-Ferritin)。将 HM2e-Ferritin 氨基酸序列按照大肠杆菌常用密码子进行优化并合成,同时单独扩增出Ferritin基因,将它们克隆至原核表达载体pET-32a。通过双酶切和测序鉴定重组质粒,将阳性质粒转入感受态细胞BL21(DE3),再经过IPTG诱导表达。通过对诱导条件、诱导温度和诱导剂浓度的优化,获得最佳的蛋白表达条件。重组蛋白经镍柱纯化后,通过SDS-PAGE鉴定,结果显示HM2e-Ferritin蛋白和Ferritin蛋白以可溶性形式存在于蛋白上清。WB鉴定表明,HM2e-Ferritin和Ferritin蛋白具有免疫反应性,HM2e-Ferritin蛋白分别具有HM2e和Ferritin的活性;利用电子透射显微镜和纳米粒度仪对重组蛋白分析,这两种蛋白均能形成纳米粒结构,Ferritin蛋白组装的颗粒大小在12～20nm,HM2e-Ferritin蛋白组装的颗粒大小在20～70nm,呈狭窄尺寸分布的纳米笼结构。结果表明,本研究通过大肠杆菌表达系统获得了具有纳米粒结构的HM2e-Ferritin蛋白,该蛋白具有生物学活性,为新型纳米疫苗的研制以及疾病诊治提供了理论基础。2.基于铁蛋白的多表位流感纳米疫苗的杆状病毒表达及鉴定参照A/Jilin/JYT-01/2018(H1N1)毒株的全基因组序列,将其HA2基因和HM2e-Ferritin基因按照密码子偏好优化合成。利用酶切连接的方法将HA2基因和HM2e-Ferritin基因分别插入pFastBacDual双载体,构建重组穿梭质粒pFBD-HA2-HM2e-Ferritin。通过重组穿梭质粒转化、蓝白斑筛选杆粒、重组杆粒转染Sf9细胞等步骤,成功拯救出重组杆状病毒。通过酶切、PCR、WB以及IFA等方法鉴定检测,结果表明成功拯救出重组杆状病毒,命名为rBV-HA2-HM2e-Ferritin。重组杆状病毒感染悬浮Sf9细胞,收取细胞悬液,通过超离、蔗糖密度梯度离心获得重组蛋白。利用电子显微镜对重组蛋白进行分析,TEM下可见约20 nm的病毒样颗粒。结果表明,通过杆状病毒系统获得包装双组分的多表位流感纳米粒,为广谱流感病毒疫苗的研发提供了新思路。3.多表位流感纳米疫苗佐剂的筛选及免疫效果评价以原核表达纯化的HM2e-Ferritin蛋白为抗原,分别与佐剂MF59、CpG、Poly:IC、Alum、MF59+CpG和MF59+Poly:IC佐剂按一定比例混合制成疫苗。将制备的疫苗分别免疫5周龄雌性C57BL/6小鼠,每次剂量为50μg/只,每隔3周免疫一次,共免疫三次,并设置阴阳性对照组、单纯抗原对照组(HM2e-Ferritin蛋白)和佐剂对照组。对于体液免疫应答,每周眼眶采取各组小鼠血,分离血清,进行IgG抗体分型和特异性IgG水平检测。结果表明,各疫苗组(含HM2e-Ferritin组)均可诱导较强的血清抗体IgG、IgG1和IgG2a应答,与对照组差异显著(P<0.001)。抗体效价结果显示,Alum和MF59+CpG疫苗组,能够显著提高特异性抗体的产生,抗体效价最高大于1:8×105,与对照组相比,统计学差异极显著(P<0.01)。对于细胞免疫应答,随机处死三免一周的小鼠,取脾脏分离淋巴细胞,进行流式分析和淋巴细胞增殖实验。结果显示,HM2e-Ferritin蛋白能够诱导T淋巴细胞免疫反应。细胞因子测定结果显示,Alum、CpG和MF59+CpG疫苗组诱导的IL-4和IFNγ反应,与PBS组相比,统计学差异显著(P<0.01)。攻毒后小鼠肺部病理变化、体重变化和存活率三项指标表明,MF59+CpG疫苗组和Poly:IC疫苗组小鼠肺部基本正常;各组体重均有下降,PBS组体重下降最快,且恢复较慢。在第7到9天时,疫苗组小鼠体重陆续恢复;MF59和MF59+CpG疫苗组小鼠存活率可达100%。综上所述,多表位流感纳米疫苗激发小鼠产生强烈的细胞免疫和体液免疫,能够有效抵抗H1N1流感病毒的感染。更多还原

【Abstract】 Influenza is an acute respiratory disease caused by influenza virus(Ⅳ)[1].Influenza outbreaks are mainly caused by subtypes of influenza A virus,and some subtypes have been spread among people.Influenza pandemic is caused by recombinant human avian influenza virus,among which influenza A H1N1,H3N2 and B viruses infect more people,showing seasonal epidemic every year,often accompanied by cough,high fever,runny nose,loss of appetite and muscle soreness.At present,avian influenza viruses H5N1 and H7N9 have broken through the interspecific barrier and have the characteristics of zoonosis,which may cause a pandemic[2].Influenza virus is prone to antigenic drift or antigenic transformation,which leads to the coexistence of multiple subtypes of influenza virus,and the cross protection between different subtypes is not good.The current influenza vaccine can not provide effective protection against the original variant influenza virus strains,so it is urgent to develop a vaccine that can prevent multiple subtypes of influenza virus at the same time.In this study,the conserved antigens HA2 and M2e of influenza virus were inserted into the N-terminal of Ferritin Nanoparticles by using the self-assembly and modifiable properties of Ferritin Nanoparticles.The multi-epitope influenza nanoparticles based on Ferritin were expressed by prokaryotic expression system and insect baculovirus expression system.Adjuvants(Alum,MF59,CpG,Poly:IC,MF59+CpG,MF59+Poly:IC).They were mixed with multi-epitope influenza nanoparticles to prepare vaccine and immunize mice.Through the immune effect,the adjuvant suitable for multi epitope influenza nano-vaccine was selected.The protective effect of each group of vaccines was evaluated by challenge experiment,which laid a foundation for the successful development of universal influenza nano vaccine.1.Prokaryotic expression and identification of multi-epitope influenza Nanovaccine based on FerritinHA2-M2e-Ferritin gene(HM2e-Ferritin for short)was formed by concatenating HA2 and M2e conservative antigen genes of H1/H3/B subtype influenza virus.The amino acid sequence of HM2e-Ferritin was optimized and synthesized according to the common codons of Escherichia coli.Meanwhile,Ferritin gene was amplified and cloned into prokaryotic expression vector pET-3 2a.The recombinant plasmids were identified by restriction enzyme digestion and sequencing.The positive plasmids were transferred into competent cells BL21(DE3)and induced by IPTG.By optimizing the induction conditions,induction temperature and inducer concentration,the best protein expression conditions were obtained.The recombinant protein was purified by nickel column and identified by SDS-PAGE.The results showed that HM2e-Ferritin protein and Ferritin protein were soluble in the protein supernatant.WB analysis showed that HM2e-Ferritin and Ferritin protein had immunoreactivity,and HM2e-Ferritin protein had HM2e and Ferritin activity,respectively.Electron transmission microscopy and nanoparticle size analyzer were used to analyze the recombinant protein.The results showed that the two proteins could form nanoparticle structure.The particle size of ferritin protein was 12～20nm,and that of HM2e-Ferritin protein was 20～70nm,which showed narrow size distribution of nano cage structure.The results showed that HM2e-Ferritin protein with nanoparticle structure was obtained by E.coli expression system.The protein has biological activity,which provides a theoretical basis for the development of new nano-vaccines and disease diagnosis and treatment.2.Baculovirus expression and identification of the multi-epitope influenza Nano-vaccine based on FerritinAccording to the whole genome sequence of a/Jilin/jyt-01/2018(H1N1)strain,HA2 gene and HM2e Ferritin gene were synthesized according to codon preference.The recombinant shuttle plasmid PFBD-HA2-HM2e-Ferritin was constructed by inserting HA2 gene and HM2e-Ferritin gene into PFastBacDual vector respectively.The recombinant baculovirus was successfully rescued through the transformation of recombinant shuttle plasmid,selection of baculovirus from blue and white spots,and transfection of recombinant baculovirus into Sf9 cells.The recombinant baculovirus named rbv-HA2-HM2e-Ferritin was successfully rescued by enzyme digestion,PCR,WB and IFA.Sf9 cells were infected with recombinant baculovirus,and the cell suspension was collected.The recombinant protein was obtained by ultracentrifugation and sucrose density gradient centrifugation.The recombinant protein was analyzed by electron microscope,and the virus like particles of about 20 nm could be seen under TEM.The results showed that the two-component multi epitope influenza nanoparticles were obtained by baculovirus system,which provided a new idea for the development of broad-spectrum influenza virus vaccine.3.Selection and evaluation of adjuvants for multi-epitope influenza NanovaccineThe purified HM2e-Ferritin protein expressed in prokaryotic cells was used as antigen.MF59,CpG,Poly:IC,Alum,MF59+CpG and MF59+Poly:IC.The adjuvants were mixed in a certain proportion to make the vaccine.Five week old female C57BL/6 mice were immunized with the prepared vaccine at a dose of 50 μg each time,once every three weeks for three times.The positive and negative control group,simple antigen control group(hm2e ferritin protein)and adjuvant control group were set up.For humoral immune response,the blood of mice in each group was collected from orbit every week,and the serum was separated for IgG antibody typing and specific IgG level detection.The results showed that all the vaccine groups(including HM2e-Ferritin group)could induce strong serum antibody IgG,IgGland IgG2a responses,with significant difference compared with the control group(P<0.001).The results of antibody titer showed that Alum and MF59+CpG vaccine groups could significantly improve the production of specific antibody,and the highest antibody titer was more than 1:8×105,which was significantly different from the control group(P<0.01).For cellular immune response,the mice immunized for one week were randomly killed,and the lymphocytes were isolated from the spleen for flow cytometry analysis and lymphocyte proliferation test.The results showed that HM2e-Ferritin could induce T lymphocyte immune response.The results of cytokine assay showed that the IL-4 and IFNy responses induced by alum,CpG and MF59+CpG vaccine groups were significantly different from those induced by PBS group(P<0.01).The lung pathological changes,body weight changes and survival rate of mice after challenge showed that MF59+CpG and Poly:IC vaccine group The lungs of mice had no significant difference;the weight of all groups decreased,and the weight of PBS group decreased the fastest and recovered slowly.On the 7 th to 9 th day,the weight of mice in the vaccine group gradually recovered,and the survival rate of mice in MF59 and MF59+CpG vaccine groups reached 100%.In conclusion,the multi epitope influenza nano vaccine can induce strong cellular and humoral immunity in mice,which can effectively resist the infection of H1N1 influenza virus.更多还原