【作者】 王善辉；

【导师】 李培锋； 王希良；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2009， 博士

【摘要】 呼吸道合胞病毒(Respiratory syncytial virus,RSV)属副黏病毒科,肺炎病毒属,为非节段性的负链RNA病毒。RSV基因组有15,222个核苷酸,主要编码10个特异性蛋白,其中膜表面的融合蛋白F和粘附蛋白G是激发机体产生保护性抗体的最主要的病毒表面抗原,是疫苗研究开发的重要内容。呼吸道合胞病毒(RSV)广泛流行于世界各地,是引起婴幼儿下呼吸道感染最常见的病原微生物之一。世界卫生组织(WHO)已将RSV疫苗列为全球疫苗计划中优先发展的疫苗之一。RSV疫苗研制虽已有40年历史,但至今尚无被批准使用的疫苗。为此,本研究利用杆状病毒昆虫表达系统表达了RSV全长的F、G蛋白,免疫Balb/c小鼠,评价了F和G全长蛋白疫苗的免疫原性和有效性。运用生物信息学软件选择F205～223,255～278,G142～204位氨基酸作为研究对象,表达并纯化了F-G融合表位蛋白。该研究为进一步研制适合我国的RSV疫苗奠定了试验基础。RSV F和G全长重组蛋白疫苗的质粒构建、表达和纯化目的表达并纯化RSV Long株F和G全长蛋白。方法采用反转录聚合酶链反应(RT-PCR)克隆了RSV A型Long株的全长F和G基因,构建了F-pFastHT A和G-pFastHT A质粒。将阳性重组质粒分别转化DH10Bac感受态细胞,获得重组杆状病毒质粒F-Bacmid和G-Bacmid。将其转染昆虫细胞,成功包装了含有目的片段的重组杆状病毒,采用Ni2+离子亲和层析柱纯化了接毒细胞的表达产物。进行SDS-PAGE电泳、间接免疫荧光和Western-blot试验鉴定重组蛋白。结果目的蛋白F和G在昆虫细胞中有特异性表达, Ni2+离子亲和层析柱能够很好的吸附重组目的蛋白。结论成功克隆了RSV F和G基因,并在Sf9昆虫细胞中获得表达。Ni2+离子亲和层析法纯化目的蛋白的纯度达85%以上,可用于免疫Balb/c小鼠。RSV F和G全长重组蛋白和表位肽疫苗免疫效果的研究目的检测RSV F和G蛋白候选疫苗的免疫原性和安全性。方法选用Mf59分别与F、G蛋白配制疫苗滴鼻免疫Balb/c小鼠;弗氏佐剂与F和G蛋白配制疫苗肌肉注射免疫Balb/c小鼠做为对照。分别采用间接ELISA法和微量中和试验检测免疫鼠血清特异性IgG和中和抗体效价;间接ELISA法检测免疫鼠肺、鼻灌洗液特异性IgG和SIgA抗体效价;MTT法检测免疫鼠脾淋巴细胞增殖;ELISPOT法检测免疫鼠脾脏淋巴细胞γ-IFN、IL-2、IL-4分泌。结果经杆状病毒昆虫表达系统表达的RSV F和G全长蛋白在Balb/c小鼠体内诱导产生了高滴度的血清IgG抗体、中和抗体和肺局部抗体,其中F蛋白疫苗诱导产生了平衡的IgG1/IgG2a。结论RSV F蛋白可刺激机体产生体液免疫和细胞免疫;G蛋白疫苗只刺激机体产生体液免疫,并引起Th2优势应答。RSV F-G表位肽疫苗的质粒构建、表达及纯化目的选择能同时刺激机体产生细胞免疫和体液免疫并诱导平衡的Th1/Th2应答的RSV表位蛋白。方法运用生物信息学软件预测F和G蛋白的空间结构,并查看资料寻找F和G蛋白的抗原表位。选择F205～223,255～278,G142～204位氨基酸作为研究对象,合成编码这两段表位的基因,采用重叠PCR将编码F和G两段表位的基因连接,杆状病毒昆虫表达系统表达F-G融合表位肽蛋白,Ni2+亲和层析纯化目的蛋白。结果融合表位蛋白F-G在昆虫细胞中有特异性表达, Ni2+离子亲和层析柱能够很好的吸附重组目的蛋白。结论成功克隆了RSV F-G融合表位基因,并在Sf9昆虫细胞中获得表达。Ni2+离子亲和层析法纯化目的蛋白的纯度达85%以上。更多还原

【Abstract】 Respiratory syncytial virus (RSV) is a kind of non-segmental RNA virus of negative strand, and it is classified as a member of the genus Pneumonia virus in the family of Paramyxoviridae. RSV genome has 15,222 nucleotides mainly coding 10 specific proteins, the fusion protein F on membrane surface and adhesion protein G, as the important aspect of research and development in vaccination, are the mainly surface antigens of RSV to induce the production of protective antibodies. RSV is one of the most common pathogen which cause the lower respiratory tract infection called asthmatic suffocating pneumonia(ASP) of newborns and infant widespread over the world.As the major respiratory pathogen in China, it makes ASP outbreak in every years.So far, RSV infection is still the primary cause of hospitalization of the infants so that the production of RSV Vaccines had been included in the global plans by WHO as one of the priority products. There is no licensed RSV vaccine after 40 years development. In this study, RSV fusion protein F and adhesion protein G have been successfully produced as RSV subunit vaccine candidate with baculovirus-Insect expression system. The immunogenicity and dffectivity of the RSV subunit vaccine candidate have been evaluated after inoculation in Balb/c mice. Amino acids of 205-223 and 255-278 from F ptotein, and 142-204 from G protein were selected to be epitopes analyzed by bioinformatic software. Epitope fusion protein of F and G have been expressed and purified successfully. It is helpful for further devolepment of RSV subunit vaccine in China.Recombinant plasmid construction, expression and purification of RSV complete F protein and G protein objective expression and purification of F and G gene of RSV (A type, Long strain). Methods F and G full gene of RSV (A type, Long strain) were amplified by RT-PCR, and the plasmid of F-pFastHT A and G-pFastHT A were constructed.The positive purified recombinant plasimid was transformed into DH10Bac E. coli for transposition into the bacmid to get F-Bacmid and G-Bacmid, respectively. The positive purified recombinant bacmids have been transfected into insect cell for successful virus packing. Product of the transfected cells were purified by Ni2+ Ion affinity chromatography and the expression of protein were detected by SDS-PAGE assay, Indirect immunofluorescence and and western-blot assay. Results The interest protein ( F and G) were expressed of specificity and absorbed efficiently by the Ni2+ affinity chromatograph column.Conclusion F gene and G gene were cloned successfully and proteins were expressed in insect cells. The purity was up to 85% by Ni2+ Ion affinity chromatography purification that it was fit for immunization to Balb/c mice.Evaluation of immune effect of recombinant protein of complete F protein and G protein and RSV subunit vaccine candidate Objective to exam the immunogenicity and safety of the vaccine candidates of F and G protein of RSV. Methods F protein and G protein were immuned intranasally to Balb/c mice with Mf59 respectively as vaccine candidates and intramuscularly with Freund’s adjuvant respectively as control. The specific IgG and neutralizing antibody titers of immune mouse serum were determined Separately by indirect ELISA and micro neutralization test; the titers of specific IgG and IgA of lung lavage fluid and nasal lavage fluid were detected by indirect ELISA in immune mouse; multiplication of splenic lymphocyte was detected by MTT assay; secretion ofγ-IFN,IL-2 and IL-4 were detected by ELISPOT assay. Results Serum IgG, neutralizing antibodies and lung local antibodies of high titers were produced in Balb/c mice by induction F protein and G protein expressed in insect baculovirus expression system. Balanced IgG1/IgG2a was induced by F protein also. Conclusion F protein could induce the humoral immunity and cell-mediated immunity, G protein only induce humoral immunity and the advantage response of Th2 cells.Recombinant plasmid construction, expression and purification of RSV F-G epitope fusion protein Objective Choosing the protein epitope that could induce humoral immunity and cell-mediated immunity simultaneously and induce the balanced Th1/Th2 response also.Methods The spatial structure of F and G protein were analyzed and forecasted and epitopes of F and G protein was found by using software of bioinformatics.205aa-223aa and 255aa-278aa of F protein and 142aa-204aa of G protein were selected and their coding gene of F and G epitope were synthesized and linked with G2SG2 by overlapping PCR. Then F-G fusion epitope protein was expressed in insect cells and purified by Ni2+ affinity chromatograph. Results Specific recombinant fusion epitope protein F-G was expressed in insect cells, and absorbed efficiently by the Ni2+ affinity chromatograph column for purification. Conclusion RSV fusion epitope gene was cloned successfully and the fusion epitope protein F-G was expressed in Sf9 insect cells. The putity of the inteset fusion protein was up to 85% by Ni2+ Ion affinity chromatography.更多还原