表达狂犬病毒G蛋白的重组新城疫病毒接种牛羊的免疫原性研究
发布时间：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兽共患病,出现临床症状后的死亡率几乎为100%。RV为弹状病毒科狂犬病毒属,为单股不分节负链RNA病毒。它主要编码N、P、M、G、L五种结构蛋白,G蛋白是RV的主要抗原蛋白,刺激机体可产生相应中和抗体并诱导细胞免疫。新城疫病毒(Newcastle Disease Virus,NDV)同样为不分节段的单股负链RNA病毒。随着反向遗传学技术的发展NDV被开发成一种安全有效的活疫苗载体。NDV作为活载体有多方面的优势:首先,NDV可以通过多种途径免疫。甚至是呼吸道途径免疫,能够引起有效的黏膜免疫、细胞免疫和体液免疫;其次NDV不会整合到宿主基因组中,生物安全性良好;最后,接种鸡胚的生产模式极大的简化了疫苗生产的程序,方便质量控制,不使用胎牛血清大幅度地降低了疫苗生产成本。我们实验室利用反向遗传操作技术,以NDV La Sota疫苗株做为表达载体(r La Sota),将RV的G蛋白插入到NDV的P基因和M基因之间构建出表达RV的重组NDV(r L-RVG)。结果显示RV G蛋白稳定高效表达,动物实验如犬、猫和老鼠注射重组疫苗r L-RVG后能够产生大量针对RV的保护性中和抗体。r La Sota作为低致病性的NDV毒株,无TPCK存在的情况下,在BHK-21细胞上培养时它能够感染单个细胞但不能从初次感染细胞扩散到相邻细胞。间接免疫荧光结果显示感染r L-RVG后24小时到96小时,r L-RVG能够从最初感染的细胞扩散到周围的邻近细胞,逐渐形成类似RV形成的病毒蚀斑。r L-RVG体现出与亲本r La Sota不同的扩散方式并未增加其对小鼠和犬、猫等实验动物的致病性,因此我们更加关注重组疫苗r L-RVG对于其它类型的哺乳动物的安全性和有效性。我们对2011-2013年间内蒙古地区收集的疑似狂犬病动物脑组织进行直接免疫荧光和RT-PCR鉴定,并根据N基因部分序列进行遗传演化分析,确定内蒙古地区的狂犬病流行毒株主要为两个来源:本地流行株和蒙古,俄罗斯传入的外来毒株。鉴于内蒙地区奶牛和绵羊等家养动物狂犬病病例的数量逐年增加,本实验中,我们评估重组病毒r L-RVG在奶牛和羊上应用的可行性。我们在MDBK细胞上比较了r L-RVG与亲本株r La Sota对牛干扰素的敏感性,以及r L-RVG诱导MDBK细胞和LT细胞产生干扰素的能力。抗病毒实验结果证明,r L-RVG对于干扰素高度敏感,与其亲本株r La Sota一致。但在病毒剂量和作用时间一致的情况下,r L-RVG诱导干扰素的能力显著高于亲本株r La Sota。以109EID50和108 EID50的大剂量肌肉接种牛和绵羊,r L-RVG与亲本株r La Sota一样并未表现出任何临床症状和生长发育异常。静脉血抗RV中和实验证明,r L-RVG能够诱导显著的保护性中和抗体,并于二次加强免疫后呈现快速上升,而牛在二次加强免疫后108 EID50低剂量组的中和抗体显著高于109EID50接种的高剂量组。109EID50剂量接种绵羊诱导的中和抗体水平高于108 EID50低剂量组的中和抗体。经过两次免疫,牛和绵羊体内的保护性中和抗体持续存在,在免疫后的第12个月中和抗体滴度仍然高于0.5IU的强毒攻击保护性阈值。结果说明,重组活疫苗r L-RVG对于牛和绵羊不具有致病性,并能有效诱导高水平中和抗体,具有进一步应用的潜力。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
，
本文编号：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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
，
本文编号：2188623
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