脊髓灰质炎(Poliomyelitis，以下简称脊灰)是由脊髓灰质炎病毒(Poliomyelitis Virus，PV)感染引起、危害极大的急性传染病，尚无有效治疗方法，只能通过疫苗预防。口服脊灰减毒活疫苗(Oral Poliomyelitis Vaccine，OPV)因接种方便，成本相对较低而一度被广泛使用，但可能引起脊灰疫苗相关麻痹病例(Vaccine-Associated Paralytic Poliomyelitis ，VAPP)和疫苗衍生株脊灰病毒(Vaccine-Derived Poliovirus，VDPV)病例，灭活脊灰疫苗(Inactivated poliomyelitis vaccine，IPV)能有效避免VAPP和VDPV，采用IPV取代OPV是《全球消灭脊灰战略终结计划》的要求。而在全世界消灭脊灰后，采用野毒株(Salk)生产IPV对生物安全水平要求更加严格，因此WHO鼓励疫苗厂家研发Sabin株脊灰灭活疫苗(Inactivated Poliomyelitis Vaccine，Sabin strain，sIPV)。在研发sIPV过程中，Sabin株毒种的遗传稳定性极其重要。本研究通过检测PV感染性滴度、D抗原含量以及PV全基因组Sanger测序的方法来检测制备的工作种子和疫苗代次病毒的遗传稳定性。

此外，生产中在对疫苗质量进行监测时，需区别通过和未通过猴体神经毒力试验(Monkeys Neurovirulence Test，MNVT)的疫苗之间的差别；而采用聚合酶链式反应和限制性内切酶酶切技术作突变分析(Mutant Analysis by PCR and Restriction Enzyme Cleavage，MAPREC)虽然可以准确地测定出一个碱基位点突变的量，但无法实现对基因组每个核苷酸的序列变化都进行监测。随着分子水平的基因检测技术平台不断发展和完善，第二代测序技术(Next Generation Sequencing，NGS)的出现，使得基因检测效率不断提高，从分子水平来监测疫苗质量更容易实现。本研究通过对Sabin株病毒NGS模板制备方法的讨论，探讨了如何去除背景核酸的干扰、低浓度样品的扩增方法选择、样品的纯化定量筛选以及质量鉴定，以及测序平台的选择等问题。

目的 1.分析sIPV毒种(亚主种子、工作种子)及疫苗代次病毒(SO + 4)、SO+5、SO+6的遗传稳定性；2. 初步建立Sabin株病毒NGS样品的预处理方法、扩增方法、纯化筛选方法以及测序平台选择等问题，以期为进一步研究Sabin株的遗传稳定性构建一种切实可行的新方法。

方法 1.比较疫苗株SabinⅠ、Ⅱ型及PfizerⅢ型亚主种子、工作种子及疫苗代次病毒、SO+5、SO+6的感染性滴度及D抗原含量的变化情况，并对上述病毒进行全基因组测序；2.采用常用的NGS样品的预处理方法、扩增方法、纯化等方法制备Sabin株病毒NGS测序模板，比较筛选适用Sabin株病毒NGS的最佳组合，并对制备的模板进行定量和质量鉴定。

结果 1.各代次Sabin株病毒滴度均维持在7.62~8.62 lgCCID50/ml，D抗原含量均维持在30~128 DU/ml。与GenBank上登录的SabinⅠ(AY184219.1)、Ⅱ型(AY184220.1)及Sabin Ⅲ型(AY184221.1)减毒株原始种子(SO)基因序列相比，Sabin株亚主种子(SO+2)、工作种子(SO+3)及疫苗代次病毒(SO+4)、SO+5、SO+6均未出现碱基突变；2. Sabin株病毒NGS模板制备的样品预处理是必要的，选用不依赖序列的单引物扩增效果最好，优化了磁珠纯化法的磁珠用量，且采用Qubit定量系统定量更精确。

结论 1.sIPV毒种及疫苗代次病毒、SO+5、SO+6的病毒全基因序列与原始种子相比，均未发生变化，毒力均一，遗传性状方面保持了较好的稳定性；2.初步建立了Sabin株病毒NGS的模板制备方法，并成功制备了Sabin株病毒NGS模板。

Poliomyelitis is widely identified to be an acute viral infectious disease with highly infectious and extremely harmful caused by poliomyelitis virus (PV). There is no effective treatment for the disease and the only way to prevent people from being infected is vaccination. Oral poliomyelitis vaccine (OPV) was widely used due to its convenient and cheap，but the vaccine-associated paralytic poliomyelitis (VAPP) and vaccine-derived poliovirus (VDPV) cases caused by OPV use have increasingly received great attention. Poliomyelitis vaccine inactivated (IPV) can avoid VAPP and VDPV，to gradually replace OPV with IPV for avoiding the VAPP and VDPV，becomes the emphasized issue in the Polio Eradication and Endgame Strategic Plan. After the eradication of polio around the world，the conventional IPV production is generally carried out facilities with bio-containment requirement more stringent. WHO encourages developing the IPV made from Sabin strain (sIPV). The genetic stability of Sabin strain is extremely important in the research of sIPV. In this study，we detected the genetic stability through PV infectious titer，D antigen and PV complete genome Sanger sequencing.

Furthermore，it’s necessary to distinct vaccine between passed and failed the monkeys neurovirulence test (MNVT) when monitoring the quality of vaccine in production，and mutant analysis by polymerase chain reaction (PCR) and restriction enzyme cleavage (MAPREC) can accurately measure the amount of a nucleotide site mutation，however， MAPREC can only monitor mutations at a few genomic loci and miss mutations at other sites that could adversely affect vaccine quality. With the development of the molecular genetic testing technology，the next generation sequencing (NGS) make it possible to sensitive detection and quantification of all mutations in the entire genome of Sabin strain. In this study， we discussed the preparation method of Sabin strains NGS template， include how to remove the interference of background nucleic acid and amplify the low concentration sample，how to purify and choose the sequencing platform.

Objective 1. To analyze the genetic stability of Sabin strain as a virus seed(sub-master and working virus seeds)for production of inactivated poliomyelitis vaccine as well as vaccine virus SO+4，SO+5 and SO+6. 2. A preliminary approach to establishing the NGS sample preparation of Sabin strains， include preprocessing， amplification， purification， screening and the sequencing platform.

Methods 1. The sub-master and working seeds of strains SabinⅠ，SabinⅡand PfizerⅢas well as vaccine virus were determined for infectious titer and D antigen content，of which the complete genomes were sequenced. 2. Compared the common NGS sample preprocessing method， amplification method and purification methods，then selected the best way of Sabin strain.

Results 1.The titers of Sabin strains of various passages were 7.62~8.26 lgCCID50/ml， while the D antigen contents were 30~128 DU/ml. Compared with the complete genome sequences of SabinⅠ(AY184219. 1)，SabinⅡ(AY184220. 1) and SabinⅢ(AY184221. 1)and attenuated strain of original seed(SO)in GenBank，the sub-master seed(SO+2)， working seed(SO+3)，vaccine seed virus(SO+4)，SO+5 and SO+6 of Sabin strain showed no base mutation. 2. It′s necessary that DNaseⅠfor sample pretreatment， and the best way to PCR is the sequence independent single primer amplification.

Conclusion 1.No change was observed in the complete genome sequence of Sabin strain as virus seed and the vaccine virus compared with that of original seed (SO)， while the virulence was homogeneous， indicating high genetic stability. 2. The template preparation method of Sabin strain NGS was preliminarily established.