Cathay拓扑型O型口蹄疫突变病毒的拯救及其抗原稳定性研究
发布时间：2019-04-11 18:32
【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的急性、烈性传染病,主要感染牛、羊及猪等偶蹄动物。口蹄疫流行地区,灭活疫苗免疫是防控疫情的关键措施,而灭活疫苗要发挥作用的关键,是确保FMDV颗粒的完整,即146S粒子的完整。然而146S粒子稳定性较差,温度升高或pH发生变化时会使146S分解为12S粒子,导致灭活疫苗免疫失败。于是研究者们根据146S粒子分子结构的研究成果,尝试通过氨基酸残基替换来提升FMDV的酸、热稳定性,进而为筛选候选疫苗毒株提供技术支持。本研究在先前的研究基础上,选取了疫苗种毒O/GX/09-7毒株的结构蛋白VP1的17位及VP2的93位氨基酸残基作为替换位点。利用设计的突变引物,成功扩增出含碱基突变的O/GX/09-7毒株P1区序列。成功将目的片段插入到T载体,构建8种克隆质粒,成功将目的片段插入到本实验室已构建好的FMDV载体p43-CHA90-Vector上,构建出8种重组质粒。利用8种重组质粒及感染性克隆pOZK/93转录出病毒基因组RNA并且转染进入BHK-21细胞中,成功拯救出2株病毒vOZK/93和vOS93H。对病毒vOZK/93和vOS93H及亲本毒株O/GX/09-7分别进行酸、热处理,测量处理后病毒的TCID50的值及146S粒子含量,并与空白对照进行比较,研究氨基酸替换对O/GX/09-7毒株稳定性的影响。结果显示,vOZK/93和vOS93H两株病毒的热稳定性均与O/GX/09-7毒株相似,没有明显变化。病毒vOZK/93的酸稳定性与O/GX/09-7毒株相似,但突变病毒vOS93H的酸稳定性与O/GX/09-7毒株相比,有一定的提高,初步达到试验预期目的。
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is an acute and severe infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV), which mainly affects cloven-hoofed animals such as cattle, sheep and pigs. In the epidemic areas of foot-and-mouth disease, inactivated vaccine immunization is the key measure to prevent and control the epidemic situation, and the key of inactivated vaccine to play a role is to ensure the integrity of FMDV particles, that is, the integrity of 146s particles. However, 146S particles have poor stability. When the temperature increases or pH changes, 146S particles will be decomposed into 12s particles, resulting in the inactivated vaccine immune failure. Therefore, according to the molecular structure of 146S particles, the researchers try to improve the acid and thermal stability of FMDV by amino acid residues substitution, and then provide technical support for screening candidate vaccine strains. On the basis of previous studies, the 17 site of structural protein VP1 and 93 amino acid residue of VP2 of O/GX/09-7 strain were selected as substitution sites. The P1 region of O/GX/09-7 strain containing base mutation was amplified by using the designed primers. The target fragment was successfully inserted into T vector to construct eight kinds of cloning plasmids. The target fragment was successfully inserted into the FMDV vector p43-CHA90-Vector which had been constructed in our laboratory, and eight recombinant plasmids were constructed. Eight recombinant plasmids and infectious clone pOZK/93 were used to transcribe the genomic RNA of the virus and transfect it into BHK-21 cells. Two strains of virus vOZK/93 and vOS93H. were successfully rescued. The virus vOZK/93, vOS93H and parent strain O/GX/09-7 were treated with acid and heat treatment respectively. The TCID50 value and 146s particle content of the virus were measured and compared with the blank control. The effect of amino acid substitution on the stability of O/GX/09-7 strain was studied. The results showed that the thermal stability of both vOZK/93 and vOS93H strains was similar to that of O/GX/09-7 strain, but there was no significant change. The acid stability of the virus vOZK/93 was similar to that of the O/GX/09-7 strain, but the acid stability of the mutant virus vOS93H was higher than that of the O/GX/09-7 strain.
【学位授予单位】：中国兽医药品监察所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：2456654
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is an acute and severe infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV), which mainly affects cloven-hoofed animals such as cattle, sheep and pigs. In the epidemic areas of foot-and-mouth disease, inactivated vaccine immunization is the key measure to prevent and control the epidemic situation, and the key of inactivated vaccine to play a role is to ensure the integrity of FMDV particles, that is, the integrity of 146s particles. However, 146S particles have poor stability. When the temperature increases or pH changes, 146S particles will be decomposed into 12s particles, resulting in the inactivated vaccine immune failure. Therefore, according to the molecular structure of 146S particles, the researchers try to improve the acid and thermal stability of FMDV by amino acid residues substitution, and then provide technical support for screening candidate vaccine strains. On the basis of previous studies, the 17 site of structural protein VP1 and 93 amino acid residue of VP2 of O/GX/09-7 strain were selected as substitution sites. The P1 region of O/GX/09-7 strain containing base mutation was amplified by using the designed primers. The target fragment was successfully inserted into T vector to construct eight kinds of cloning plasmids. The target fragment was successfully inserted into the FMDV vector p43-CHA90-Vector which had been constructed in our laboratory, and eight recombinant plasmids were constructed. Eight recombinant plasmids and infectious clone pOZK/93 were used to transcribe the genomic RNA of the virus and transfect it into BHK-21 cells. Two strains of virus vOZK/93 and vOS93H. were successfully rescued. The virus vOZK/93, vOS93H and parent strain O/GX/09-7 were treated with acid and heat treatment respectively. The TCID50 value and 146s particle content of the virus were measured and compared with the blank control. The effect of amino acid substitution on the stability of O/GX/09-7 strain was studied. The results showed that the thermal stability of both vOZK/93 and vOS93H strains was similar to that of O/GX/09-7 strain, but there was no significant change. The acid stability of the virus vOZK/93 was similar to that of the O/GX/09-7 strain, but the acid stability of the mutant virus vOS93H was higher than that of the O/GX/09-7 strain.
【学位授予单位】：中国兽医药品监察所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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