本文選題：呼吸道合胞病毒（RSV） + A/Puerto　； 參考：《重慶大學(xué)》2012年碩士論文

【摘要】：目的在成功構(gòu)建的三株嵌合RSV抗原表位的重組流感減毒疫苗的基礎(chǔ)上，本研究對其多項指標(biāo)進行鑒定，然后對其安全性、免疫原性及免疫保護性進行一個初步的研究，從中找到一種高效價株并探討其簡便的免疫接種途徑。 方法運用血凝、血抑實驗確定是否成功轉(zhuǎn)染出重組流感病毒；通過RT-PCR、SDS-PAGE、間接免疫熒光、電鏡觀察病毒形態(tài)等方法驗證轉(zhuǎn)染出的病毒是否正確；接種雞胚對病毒進行擴大培養(yǎng)后經(jīng)蔗糖密度梯度離心純化，TCID50方法測定其病毒滴度；通過動物實驗對其安全性、免疫原性及免疫保護性進行一個初步的研究。 結(jié)果血凝、血抑實驗測得為陽性，表明成功轉(zhuǎn)染出重組流感病毒；經(jīng)RT-PCR鑒定重組病毒基因與質(zhì)粒序列相同，SDS-PAGE電泳鑒定表明流感的主要成分都存在；間接免疫熒光(IFA)檢測表明拯救出的病毒是以A/PR/8/34為骨架的重組病毒；電鏡觀察重組病毒與流感病毒的形態(tài)特征相似；通過蔗糖密度梯度法對重組病毒進行純化后，測得RSV-FLU/NS1-F、RSV-FLU/NS1-G和RSV-FLU/NS1-F+G的病毒滴度分別為10~(-7.82)/ml、10~(-7.5)/ml、10~(-8)/ml，10~(-5)TCID_(50)的病毒經(jīng)滴鼻途徑免疫BALB/c小鼠，通過對小鼠肺，鼻，腦中病毒載量的檢測發(fā)現(xiàn)，腦中沒有檢測到病毒，，1天后鼻腔檢測不到病毒RSV-FLU/NS1-F+G，第3天時檢測不到RSV-FLU/NS1-F和RSV-FLU/NS1-G，10天后肺部檢測不到病毒，實驗中沒有小鼠死亡，這說明重組病毒在小鼠上是安全的。對BALB/c小鼠鼻腔接種免疫表明：三株重組RSV-FLU/NS1病毒都產(chǎn)生了較高的HI及一定水平的針對RSV的抗體效價，同時也產(chǎn)生了sIgA。IgG1和IgG2a的測定和ELISpot實驗對細(xì)胞因子IL-4和IFN-γ的分泌性細(xì)胞水平的檢測結(jié)果表明重組病毒可以同時激活機體的細(xì)胞和體液免疫應(yīng)答。小鼠血清中細(xì)胞因子的測定結(jié)果為IL-4和IFN-γ的分泌均比PBS組高。對病毒的免疫保護性評價結(jié)果為RSV-FLU/NS1-F和RSV-FLU/NS1-G重組病毒疫苗對保護小鼠肺損傷具一定的作用，但RSV-FLU/NS1-F+G重組病毒疫苗的保護效果不理想。 結(jié)論成功轉(zhuǎn)染出三株嵌合RSV抗原表位的重組流感減毒疫苗，經(jīng)鑒定三株減毒疫苗基因組正確。免疫效果評價結(jié)果表明三株減毒疫苗是安全的，并且能夠引起小鼠體內(nèi)的免疫應(yīng)答。其中，RSV-FLU/NS1-F和RSV-FLU/NS1-G重組病毒疫苗對保護小鼠肺損傷具一定的作用，但RSV-FLU/NS1-F+G重組病毒疫苗保護效果不理想。
[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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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