嵌合RSV抗原表位的重组流感减毒疫苗的免疫原性及免疫保护性的初步研究

发布时间：2018-05-04 14:31

本文选题：呼吸道合胞病毒（RSV） + A/Puerto　；参考：《重庆大学》2012年硕士论文

【摘要】：目的在成功构建的三株嵌合RSV抗原表位的重组流感减毒疫苗的基础上，本研究对其多项指标进行鉴定，然后对其安全性、免疫原性及免疫保护性进行一个初步的研究，从中找到一种高效价株并探讨其简便的免疫接种途径。方法运用血凝、血抑实验确定是否成功转染出重组流感病毒；通过RT-PCR、SDS-PAGE、间接免疫荧光、电镜观察病毒形态等方法验证转染出的病毒是否正确；接种鸡胚对病毒进行扩大培养后经蔗糖密度梯度离心纯化，TCID50方法测定其病毒滴度；通过动物实验对其安全性、免疫原性及免疫保护性进行一个初步的研究。结果血凝、血抑实验测得为阳性，表明成功转染出重组流感病毒；经RT-PCR鉴定重组病毒基因与质粒序列相同，SDS-PAGE电泳鉴定表明流感的主要成分都存在；间接免疫荧光(IFA)检测表明拯救出的病毒是以A/PR/8/34为骨架的重组病毒；电镜观察重组病毒与流感病毒的形态特征相似；通过蔗糖密度梯度法对重组病毒进行纯化后，测得RSV-FLU/NS1-F、RSV-FLU/NS1-G和RSV-FLU/NS1-F+G的病毒滴度分别为10~(-7.82)/ml、10~(-7.5)/ml、10~(-8)/ml，10~(-5)TCID_(50)的病毒经滴鼻途径免疫BALB/c小鼠，通过对小鼠肺，鼻，脑中病毒载量的检测发现，脑中没有检测到病毒，，1天后鼻腔检测不到病毒RSV-FLU/NS1-F+G，第3天时检测不到RSV-FLU/NS1-F和RSV-FLU/NS1-G，10天后肺部检测不到病毒，实验中没有小鼠死亡，这说明重组病毒在小鼠上是安全的。对BALB/c小鼠鼻腔接种免疫表明：三株重组RSV-FLU/NS1病毒都产生了较高的HI及一定水平的针对RSV的抗体效价，同时也产生了sIgA。IgG1和IgG2a的测定和ELISpot实验对细胞因子IL-4和IFN-γ的分泌性细胞水平的检测结果表明重组病毒可以同时激活机体的细胞和体液免疫应答。小鼠血清中细胞因子的测定结果为IL-4和IFN-γ的分泌均比PBS组高。对病毒的免疫保护性评价结果为RSV-FLU/NS1-F和RSV-FLU/NS1-G重组病毒疫苗对保护小鼠肺损伤具一定的作用，但RSV-FLU/NS1-F+G重组病毒疫苗的保护效果不理想。结论成功转染出三株嵌合RSV抗原表位的重组流感减毒疫苗，经鉴定三株减毒疫苗基因组正确。免疫效果评价结果表明三株减毒疫苗是安全的，并且能够引起小鼠体内的免疫应答。其中，RSV-FLU/NS1-F和RSV-FLU/NS1-G重组病毒疫苗对保护小鼠肺损伤具一定的作用，但RSV-FLU/NS1-F+G重组病毒疫苗保护效果不理想。
[Abstract]:Objective on the basis of the successful recombinant influenza vaccine of three strains of chimeric RSV antigen epitopes, a number of indicators were identified and a preliminary study on its safety, immunogenicity and immunity protection was carried out to find a highly effective strain and explore its simple immunization route.
Methods the recombinant influenza virus was successfully transfected with hemagglutination and blood inhibition test. The virus was verified by RT-PCR, SDS-PAGE, indirect immunofluorescence and electron microscope observation of virus morphology. The virus was purified by sucrose density gradient centrifugation after inoculated with chicken embryo and the virus drops were determined by TCID50 method. A preliminary study on its safety, immunogenicity and immune protection was carried out through animal experiments.
The results showed that the recombinant influenza virus was transfected successfully. The recombinant virus gene was the same as the plasmid sequence identified by RT-PCR. The SDS-PAGE electrophoresis identification showed that the main components of influenza were present, and the indirect immunofluorescence (IFA) detection showed that the rescued virus was a recombinant virus with A/PR/8/34 as the skeleton. The morphological characteristics of the recombinant virus were similar to that of influenza virus. After the recombinant virus was purified by the sucrose density gradient method, the virus titers of RSV-FLU/NS1-F, RSV-FLU/NS1-G and RSV-FLU/NS1-F+G were 10~ (-7.82) /ml, 10~ (-7.5) /ml, 10~ (-8) /ml. The detection of viral load in the lungs, nose and brain found that the virus was not detected in the brain, the virus RSV-FLU/NS1-F+G was not detected in the nasal cavity 1 days later, the RSV-FLU/NS1-F and RSV-FLU/NS1-G were not detected at third days, and the lungs were not detected in the lungs for 10 days, and no mice were killed in the experiment. This said that the recombinant virus was safe in mice. To the nose of BALB/c mice. Cavity immunization showed that three recombinant RSV-FLU/NS1 viruses produced high HI and a certain level of antibody titer against RSV. Meanwhile, the determination of sIgA.IgG1 and IgG2a and the detection of secretory cell levels of cytokines IL-4 and IFN- gamma in ELISpot experiments showed that the recombinant virus could activate the cells of the body simultaneously. The results of IL-4 and IFN- gamma secretion in the serum of mice were higher than that in the PBS group. The protective effect of RSV-FLU/NS1-F and RSV-FLU/NS1-G recombinant virus vaccine had a certain effect on protecting the lung injury of mice, but the protective effect of the RSV-FLU/NS1-F +G recombinant virus vaccine was not ideal.
Conclusion the recombinant influenza vaccine was successfully transfected into three chimeric RSV epitopes, and the genome of three attenuated vaccine was correctly identified. The results of the immune effect evaluation showed that three attenuated vaccines were safe and could induce immune responses in mice. The RSV-FLU/NS1-F and RSV-FLU/NS1-G recombinant virus vaccines were used to protect the lung of mice. The injury has a certain effect, but the protective effect of RSV-FLU/NS1-F+G recombinant virus vaccine is not ideal.

【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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