【作者】 张国际；

【导师】 李晓眠；

【作者基本信息】 天津医科大学 ， 病原生物学， 2007， 博士

【摘要】 副流感病毒可引起一系列的呼吸道疾病,哮吼(Croup)是副流感病毒感染的典型症状,其中以副流感病毒1型(hPIV1)感染最为明显,如果感染hPIV1的婴幼儿不及时入院治疗,患儿将出现严重的呼吸道感染症状,甚至危及生命,同时,婴幼儿人群hPIV1患病率将明显升高。不仅如此，它又可以累及成人。仙台病毒(Sendai Virus, SeV),也称鼠源副流感病毒1型,是负单链RNA病毒,仙台病毒可以引起多种啮齿类动物呼吸道疾病,甚至爆发致死性肺炎。仙台病毒感染宿主细胞由2种包膜糖蛋白HN和F介导,两种糖蛋白是主要保护性抗原。一项研究发现仙台病毒(鼠源副流感病毒1型)减毒活疫苗能够保护非洲绿猴免受人源副流感病毒1型的攻击,为仙台病毒抗原成分保护婴幼儿免受副流感病毒1型感染提供了实验依据,但hPIV1或SeV的F蛋白以及仙台病毒两种包膜糖蛋白HN和F的联合免疫效果的研究,文献未见报道。8年前从爆发急性呼吸道疾病的普通棉耳狨猴群体中分离出一株高血凝效价的病毒株,初步确认为仙台病毒,并将其命名为仙台病毒tianjin株,后更名为副粘病毒Tianjin株,近期研究提示Tianjin株为仙台病毒新的基因型。因此,我们以此株病毒构建HN基因和F基因真核表达质粒,单独或联合免疫幼鼠,观察它们在幼鼠体内的免疫应答和免疫保护作用,评价构建的真核表达质粒及其免疫方法的效果。使用RT-PCR扩增病毒HN基因的全部编码区,HindⅢ和XbaⅠ双酶切后,定向插入pcDNA3真核表达载体,构建副粘病毒Tianjin株HN真核表达质粒；以病毒cDNA为模板,PCR扩增副粘病毒Tianjin株F基因的全部编码区,F基因片段与T载体连接,构建过渡质粒,EcoRⅠ和NotⅠ双酶切过渡质粒后,酶切片段定向插入pcDNA3真核表达载体,构建副粘病毒Tianjin株F真核表达质粒。两种真核表达质粒经PCR、酶切和测序鉴定,证明构建质粒结构正确。构建成功的HN基因和F基因真核表达质粒为下一步的体外表达和动物免疫实验奠定了基础。成功构建的HN基因和F基因真核表达质粒分别命名为pcDNA.HN和pcDNA.F,质粒经去内毒素处理后,借助阳离子脂质体转染COS7细胞,进行瞬时表达,应用RT-PCR、免疫荧光技术和ELISA方法检测重组子在COS7细胞的转录和翻译。RT-PCR结果提示pcDNA.HN和pcDNA.F可以在COS7细胞转录；间接免疫荧光结果显示转染细胞可见较弱的黄绿色荧光物质,而空质粒转染的COS7细胞未见有荧光物质出现；原位ELISA和使用细胞裂解物作为抗原的ELISA检测结果一致,并且均提示COS7细胞可以表达抗原蛋白。证明表达成功的HN基因和F基因真核表达质粒为动物免疫实验奠定了基础。选取4-6周龄ICR幼鼠,肌内注射布比卡因预处理24h后,纯化的去内毒素的真核表达质粒pcDNA.HN和pcDNA.F单独或联合经肌内途径免疫幼鼠,动态观察幼鼠的免疫应答水平；4周后经鼻腔同株病毒攻击小鼠进行免疫保护实验,观察小鼠的存活情况、靶器官的变化和免疫应答水平。以灭活的副粘病毒Tianjin株加Al(OH)3佐剂作为阳性对照、空质粒pcDNA3和生理盐水作为阴性对照,通过小鼠存活率、脾脏和胸腺脏器比、肺组织病理变化、血凝抑制(HI)抗体、溶血抑制(HLI)抗体、B细胞和T细胞增殖反应、血清IgM和IgG抗体、细胞因子IFN-γ,IL-2和IL-4等指标评价副粘病毒Tianjin株HN基因和F基因真核表达质粒单独和联合使用的免疫效果。实验结果显示副粘病毒Tianjin株HN基因和F基因真核表达质粒可以很好地诱发幼鼠的体液免疫和细胞免疫应答,能够保护小鼠免受随后同株病毒的攻击；并且多项指标(B细胞刺激指数、T细胞刺激指数、血清IgM和IgG水平)显示副粘病毒Tianjin株HN基因真核表达质粒的免疫效果好于F基因真核表达质粒,同时小鼠存活率、肺脏病理学变化、HI抗体、HLI抗体、B细胞刺激指数、T细胞刺激指数,血清IgG水平、血清IFN-γ和IL-2水平等指标均显示联合免疫效果好于单独免疫效果。小鼠存活率、肺脏病理学变化、B细胞刺激指数、T细胞刺激指数、血清IgG抗体、血清IFN-γ和工L-2水平支持灭活病毒组幼鼠的免疫应答和免疫保护作用好于真核表达质粒单独使用的免疫效果,而灭活病毒组与联合质粒组的免疫效果之间的比较仍需进一步研究。实验结果显示真核表达质粒pcDNA.HN和pcDNA.F联合免疫在预防幼鼠同株病毒呼吸道感染的效果明显,为副粘病毒Tianjin株包膜HN和F蛋白预防婴幼儿副流感病毒感染提供了实验动物依据。在现有的免疫方法不能有效预防婴幼儿副流感病毒感染的情况下,副粘病毒Tianjin株主要保护性抗原HN和F的DNA免疫不失为一种备选方案。更多还原

【Abstract】 Parainfluenza virus has been known to be pathogen in a series of respiratory tract infections, among which croup is a typical sign of hPIV1 infection in children, if the infants and toddlers suffering from the hPIV1 infections can not be hospitalized and treated timely, not only will the infants have severe symptoms of respiratory tract infections, even endanger their lives, but the morbidity also appears to be significantly increased. In addition, hPIV1 infection also involved in adult patients. Sendai virus, also known as murine PIV1, a (-)ss RNA virus, gives rise to respiratory tract infections in many rodents, even results in fatal pneumonia among mice. The first step in the infection process is mediated by two kinds of envelope glycoproteins HN and F of the virion, which are main protective antigens as well. A study showed attenuated Sendai virus can protect Africa green monkey from challenge with hPIV1, it provides with experimental evidence that antigenic components of Sendai virus could protect infants human being from hPIV1 infections. However the research on immunization of F glycoprotein of hPIV1 or SeV and of combination of HN and F glycoproteins of Sendai virus was poorly understood, a strain of virus possessing high HA titer was isolated from common cotton-eared marmosets suffering from acute respiratory tract infection, it was verified primarily as a strain of Sendai virus so that Sendai virus, tianjin strain was then designated at the first place and Paramyxovirus, Tianjin strain was renamed later, current research shows the virus could be a new genotype of Sendai virus. therefore, eukaryotic expression vectors harbouring HN and F gene of the Sendai virus were constructed and given solely and jointly to infant mice, its immune response and immunological protection against subsequent infection were observed in order to evaluate these constructs and their administration approaches. HN coding region of the viral genome was amplified using RT-PCR, after the PCR product was digested by both HindⅢand Xba I, it was inserted unilaterally into downstream of Pcmv of pcDNA3 and eukaryotic expression vector harbouring HN gene of Paramyxovirus, Tianjin strain was then constructed. On the other hand, F coding region of the genome was amplified by PCR using its cDNA as template, and then F PCR products and T vector was ligated together to construct transitional plasmid which makes it easier for the plasmid to be digested by both EcoR I and Not I, the enzyme-digested fragment was later inserted to pcDNA3 as it did the case of HN. Both constructs were confirmed by such protocols as PCR, RE digestion and sequencing.Successfully-constructed eukaryotic expression vectors were designated as pcDNA.HN and pcDNA.F respectively, both constructs transfected’COS7 cell for instant expression with the aid of cation liposome after being de-endotoxinated, their transcription and translation were then checked by protocols such as RT-PCR, IFA and ELISA. RT-PCR results showed both pcDNA.HN and pcDNA.F can transcript mRNA in COS7 cell. In addition, IFA showed that pale greenyellow fluorescence substance was seen in the construct-transfected cells whereas no fluorescence stain in the pcDNA3-transfected cells. Furthermore, both ELISA in situ and ELISA of cell lysate produced similar results that both construct can trigger translation in COS7 cell.De-endotoxinated recombinant pcDNA.HN and pcDNA.F were administrated solely and jointly via intramuscular route in 4-6 week old infant ICR mice after pre-treated the mice with bupivacaine, their immunization was monitored on a regular weekly basis. The immunological protection test was carried out by challenging the mice population with the same strain of the virus after the mice had been inoculated three times for 4 weeks, and its protective outcomes were examined by variables such as survival rate, target organ responses and immune response, that is, variables such as survival rates, spleen index, thymus index, pathology of lung, sera HI titer, sera HLI titer, SI of lymphocyte B&T, sera IgM&IgG level, sera IFN-γ, IL-2 and IL-4 level were employed for immunological evaluation, compared to inactivated Paramyxovirus, Tianjin strain as positive control, and to both pcDNA3 and NS as negative control. the experiment results showed combination of pcDNA.HN and pcDNA.F can efficiently induce humoral immunity and cellular mediated immunity, so that these recombinants can protect the mice against subsequent challenge with the same strain of the virus. At the same time, many variables including SI of lymphocyte B&T, sera IgM&IgG level manifested the immune response induced by pcDNA.HN seemed to be better than that of pcDNA.F, most variables such as survival rates, pathology of lung, sera HI and HLI titer, SI of lymphocyte B&T, sera IgG level, sera IFN-y and IL-2 level indicated that combination approach seemed to be superior to that of sole one. Variables such as survival rate, pathology of lung, SI of lymphocyte B&T, sera IgG level, sera IFN-y and IL-2 level support an idea that immunization induced by the inactivated’virus appeared to be more effective than that of pcDNA.HN or pcDNA.F, nevertheless, the immunization compared between the construct combination and the inactivated virus needs to be further confirmed.In summary, combination approach of pcDNA.HN and pcDNA.F had satisfactory performance on preventing infant mice from respiratory tract infections by Paramyxovirus, Tianjin strain, it laid experimental foundation upon which the viral envelope glycoproteins HN and F prevent infant from hPIV1-related infections. Under such circumstance that current approach has no effective preventive intervention against hPIV1 infections in infant, should the main protective antigens HN and F’s DNA immunization be considered as a alternative to prevent hPIV1 infections.更多还原