表達(dá)狂犬病毒G蛋白的重組新城疫病毒接種牛羊的免疫原性研究
發(fā)布時(shí)間：2018-08-17 19:13
【摘要】：狂犬病(Rabies)是由狂犬病病毒(Rabies Virus,RV)引起的高致病性的人獸共患病,出現(xiàn)臨床癥狀后的死亡率幾乎為100%。RV為彈狀病毒科狂犬病毒屬,為單股不分節(jié)負(fù)鏈RNA病毒。它主要編碼N、P、M、G、L五種結(jié)構(gòu)蛋白,G蛋白是RV的主要抗原蛋白,刺激機(jī)體可產(chǎn)生相應(yīng)中和抗體并誘導(dǎo)細(xì)胞免疫。新城疫病毒(Newcastle Disease Virus,NDV)同樣為不分節(jié)段的單股負(fù)鏈RNA病毒。隨著反向遺傳學(xué)技術(shù)的發(fā)展NDV被開發(fā)成一種安全有效的活疫苗載體。NDV作為活載體有多方面的優(yōu)勢:首先,NDV可以通過多種途徑免疫。甚至是呼吸道途徑免疫,能夠引起有效的黏膜免疫、細(xì)胞免疫和體液免疫;其次NDV不會整合到宿主基因組中,生物安全性良好;最后,接種雞胚的生產(chǎn)模式極大的簡化了疫苗生產(chǎn)的程序,方便質(zhì)量控制,不使用胎牛血清大幅度地降低了疫苗生產(chǎn)成本。我們實(shí)驗(yàn)室利用反向遺傳操作技術(shù),以NDV La Sota疫苗株做為表達(dá)載體(r La Sota),將RV的G蛋白插入到NDV的P基因和M基因之間構(gòu)建出表達(dá)RV的重組NDV(r L-RVG)。結(jié)果顯示RV G蛋白穩(wěn)定高效表達(dá),動物實(shí)驗(yàn)如犬、貓和老鼠注射重組疫苗r L-RVG后能夠產(chǎn)生大量針對RV的保護(hù)性中和抗體。r La Sota作為低致病性的NDV毒株,無TPCK存在的情況下,在BHK-21細(xì)胞上培養(yǎng)時(shí)它能夠感染單個(gè)細(xì)胞但不能從初次感染細(xì)胞擴(kuò)散到相鄰細(xì)胞。間接免疫熒光結(jié)果顯示感染r L-RVG后24小時(shí)到96小時(shí),r L-RVG能夠從最初感染的細(xì)胞擴(kuò)散到周圍的鄰近細(xì)胞,逐漸形成類似RV形成的病毒蝕斑。r L-RVG體現(xiàn)出與親本r La Sota不同的擴(kuò)散方式并未增加其對小鼠和犬、貓等實(shí)驗(yàn)動物的致病性,因此我們更加關(guān)注重組疫苗r L-RVG對于其它類型的哺乳動物的安全性和有效性。我們對2011-2013年間內(nèi)蒙古地區(qū)收集的疑似狂犬病動物腦組織進(jìn)行直接免疫熒光和RT-PCR鑒定,并根據(jù)N基因部分序列進(jìn)行遺傳演化分析,確定內(nèi)蒙古地區(qū)的狂犬病流行毒株主要為兩個(gè)來源:本地流行株和蒙古,俄羅斯傳入的外來毒株。鑒于內(nèi)蒙地區(qū)奶牛和綿羊等家養(yǎng)動物狂犬病病例的數(shù)量逐年增加,本實(shí)驗(yàn)中,我們評估重組病毒r L-RVG在奶牛和羊上應(yīng)用的可行性。我們在MDBK細(xì)胞上比較了r L-RVG與親本株r La Sota對牛干擾素的敏感性,以及r L-RVG誘導(dǎo)MDBK細(xì)胞和LT細(xì)胞產(chǎn)生干擾素的能力�？共《緦�(shí)驗(yàn)結(jié)果證明,r L-RVG對于干擾素高度敏感,與其親本株r La Sota一致。但在病毒劑量和作用時(shí)間一致的情況下,r L-RVG誘導(dǎo)干擾素的能力顯著高于親本株r La Sota。以109EID50和108 EID50的大劑量肌肉接種牛和綿羊,r L-RVG與親本株r La Sota一樣并未表現(xiàn)出任何臨床癥狀和生長發(fā)育異常。靜脈血抗RV中和實(shí)驗(yàn)證明,r L-RVG能夠誘導(dǎo)顯著的保護(hù)性中和抗體,并于二次加強(qiáng)免疫后呈現(xiàn)快速上升,而牛在二次加強(qiáng)免疫后108 EID50低劑量組的中和抗體顯著高于109EID50接種的高劑量組。109EID50劑量接種綿羊誘導(dǎo)的中和抗體水平高于108 EID50低劑量組的中和抗體。經(jīng)過兩次免疫,牛和綿羊體內(nèi)的保護(hù)性中和抗體持續(xù)存在,在免疫后的第12個(gè)月中和抗體滴度仍然高于0.5IU的強(qiáng)毒攻擊保護(hù)性閾值。結(jié)果說明,重組活疫苗r L-RVG對于牛和綿羊不具有致病性,并能有效誘導(dǎo)高水平中和抗體,具有進(jìn)一步應(yīng)用的潛力。
[Abstract]:Rabies is a highly pathogenic zoonosis caused by rabies virus (RV). The mortality rate after clinical symptoms is almost 100%. RV belongs to the genus Rabies of the Ballivirus family, which is a single stranded, unsegmented negative-stranded RNA virus. With the development of reverse genetics, NDV has been developed into a safe and effective live vaccine vector. NDV has many advantages as a living vector: first, NDV can be used in many ways. Even respiratory immunity can induce effective mucosal, cellular and humoral immunity; secondly, NDV is not integrated into the host genome and has good biosafety; finally, the production mode of inoculated chicken embryos greatly simplifies the procedure of vaccine production, facilitates quality control, and greatly reduces the use of fetal bovine serum. Using the NDV La Sota vaccine strain as the expression vector (r La Sota), we constructed the recombinant NDV (r L-RVG) expressing RV by inserting the G protein of RV into the P gene and M gene of NDV. The recombinant vaccine R L-RVG produced a large number of protective neutralizing antibodies against RV. As a low pathogenic NDV strain, R La Sota could infect single cells but could not spread to adjacent cells when cultured on BHK-21 cells without TPCK. Indirect immunofluorescence showed that it could infect single cells 24 hours after infection. By 96 hours, R L-RVG could spread from the initially infected cells to adjacent cells and gradually form RV-like plaques. The R L-RVG showed that the different diffusion modes of R L-RVG did not increase its pathogenicity to mice, dogs, cats and other experimental animals. Therefore, we paid more attention to the recombinant vaccine R L-RVG for other classes. We identified the brain tissues of suspected rabies animals collected in Inner Mongolia from 2011 to 2013 by direct immunofluorescence and RT-PCR, and analyzed the genetic evolution according to the partial sequence of N gene. The results showed that the rabies epidemic strains in Inner Mongolia were mainly from two sources: local epidemic strains and local epidemic strains. In view of the increasing number of rabies cases in domestic animals such as cows and sheep in Inner Mongolia, we assessed the feasibility of recombinant R L-RVG in dairy cows and sheep. The ability of L-RVG to induce interferon production in MDBK and LT cells was demonstrated by antiviral experiments. The results showed that r-L-RVG was highly sensitive to interferon and consistent with its parent strain, R La Sota. However, the ability of r-L-RVG to induce interferon was significantly higher than that of the parent strain, R La Sota, at the same dosage and time of the virus. The results showed that R L-RVG could induce significant protective neutralizing antibodies in vein blood, and showed a rapid increase after the second immunization, while the bovines in the low dose group of 108 EID50 after the second immunization showed a rapid increase. The neutralizing antibody level of sheep inoculated with 109 EID50 was higher than that of 108 EID50. After two immunizations, the protective neutralizing antibody persisted in cattle and sheep, and the titer of the neutralizing antibody was still higher than that of 0.5 IU in 12 months after immunization. The results showed that the recombinant live vaccine R L-RVG was not pathogenic to cattle and sheep, and could effectively induce high level neutralizing antibodies, which had potential for further application.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
，
本文編號：2188623
[Abstract]:Rabies is a highly pathogenic zoonosis caused by rabies virus (RV). The mortality rate after clinical symptoms is almost 100%. RV belongs to the genus Rabies of the Ballivirus family, which is a single stranded, unsegmented negative-stranded RNA virus. With the development of reverse genetics, NDV has been developed into a safe and effective live vaccine vector. NDV has many advantages as a living vector: first, NDV can be used in many ways. Even respiratory immunity can induce effective mucosal, cellular and humoral immunity; secondly, NDV is not integrated into the host genome and has good biosafety; finally, the production mode of inoculated chicken embryos greatly simplifies the procedure of vaccine production, facilitates quality control, and greatly reduces the use of fetal bovine serum. Using the NDV La Sota vaccine strain as the expression vector (r La Sota), we constructed the recombinant NDV (r L-RVG) expressing RV by inserting the G protein of RV into the P gene and M gene of NDV. The recombinant vaccine R L-RVG produced a large number of protective neutralizing antibodies against RV. As a low pathogenic NDV strain, R La Sota could infect single cells but could not spread to adjacent cells when cultured on BHK-21 cells without TPCK. Indirect immunofluorescence showed that it could infect single cells 24 hours after infection. By 96 hours, R L-RVG could spread from the initially infected cells to adjacent cells and gradually form RV-like plaques. The R L-RVG showed that the different diffusion modes of R L-RVG did not increase its pathogenicity to mice, dogs, cats and other experimental animals. Therefore, we paid more attention to the recombinant vaccine R L-RVG for other classes. We identified the brain tissues of suspected rabies animals collected in Inner Mongolia from 2011 to 2013 by direct immunofluorescence and RT-PCR, and analyzed the genetic evolution according to the partial sequence of N gene. The results showed that the rabies epidemic strains in Inner Mongolia were mainly from two sources: local epidemic strains and local epidemic strains. In view of the increasing number of rabies cases in domestic animals such as cows and sheep in Inner Mongolia, we assessed the feasibility of recombinant R L-RVG in dairy cows and sheep. The ability of L-RVG to induce interferon production in MDBK and LT cells was demonstrated by antiviral experiments. The results showed that r-L-RVG was highly sensitive to interferon and consistent with its parent strain, R La Sota. However, the ability of r-L-RVG to induce interferon was significantly higher than that of the parent strain, R La Sota, at the same dosage and time of the virus. The results showed that R L-RVG could induce significant protective neutralizing antibodies in vein blood, and showed a rapid increase after the second immunization, while the bovines in the low dose group of 108 EID50 after the second immunization showed a rapid increase. The neutralizing antibody level of sheep inoculated with 109 EID50 was higher than that of 108 EID50. After two immunizations, the protective neutralizing antibody persisted in cattle and sheep, and the titer of the neutralizing antibody was still higher than that of 0.5 IU in 12 months after immunization. The results showed that the recombinant live vaccine R L-RVG was not pathogenic to cattle and sheep, and could effectively induce high level neutralizing antibodies, which had potential for further application.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
，
本文編號：2188623
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