基于HCMV-gB抗原的疫苗分子设计及其免疫效果评价
发布时间：2019-02-19 20:38
【摘要】：人巨细胞病毒(Human cytomegalovirus,HCMV)属于疱疹病毒科β亚科病毒,其感染对胎儿和免疫功能低下者危害严重。接种疫苗可能是某些高危人群预防和控制HCMV感染的有效措施。基于病毒表面糖蛋白gB的基因工程疫苗在孕期妇女与移植患者的二期临床试验中显示了良好的保护效果。为了进一步增强gB疫苗的免疫保护效果,本研究对新型gB候选疫苗抗原进行了分子设计和免疫效果评价。 根据已经发表的HSV-1-gB的晶体结构,通过SWISS-Prot蛋白质晶体数据库在线模拟得到了gB的三维结构。模拟结果表明,已经发现的所有三个中和表位区AD-1、AD-2和DLD都位于gB分子的膜远端球状结构域。根据三维模拟结构和维持球状结构域构象的原则,本研究设计了gB1和gB2两个新型候选疫苗抗原。通过重组PCR扩增出编码gB1和gB2的融合基因片段,将其定向克隆到哺乳动物细胞表达载体pCI-neo中,用贴壁培养的293FT细胞进行蛋白的瞬时表达,并通过镍柱亲和层析纯化目的蛋白。免疫印迹试验表明纯化的两个重组目的蛋白具有抗原性。 以CpG-ODN + Al(OH)3作为gB1和gB2的免疫佐剂,通过两针肌肉注射免疫BALB/c小鼠,在五周内快速制备获得了两个重组蛋白的高效价免疫血清,经ELISA检测抗原特异IgG抗体滴度分别为1:6400～1:51200和1:25600～1:51200。用免疫血清对表达GFP的HCMV-VR1578病毒进行微量中和试验,结果表明:无补体存在时gB1和gB2免疫血清50%中和滴度分别为1:51.8和1:24.1,加入家兔补体后分别为1:744和1:264。 综上所述,本研究成功地设计并验证了两个具有良好应用前景的新型HCMV-gB候选疫苗抗原,为进一步研究HCMV亚单位疫苗奠定了基础。
[Abstract]:Human cytomegalovirus (Human cytomegalovirus,HCMV) belongs to 尾 subfamily of herpesvirus family. Vaccination may be an effective measure to prevent and control HCMV infection in some high-risk population. The genetic engineering vaccine based on virus surface glycoprotein (gB) has shown good protective effect in the second phase clinical trial of pregnant women and transplant patients. In order to further enhance the immune protection of gB vaccine, molecular design and immunological evaluation of novel gB vaccine antigen were carried out in this study. According to the published crystal structure of HSV-1-gB, the 3D structure of gB was obtained by on-line simulation of SWISS-Prot protein crystal database. The simulation results show that all three neutralizing epitopes, AD-1,AD-2 and DLD, are located in the spherical domain at the distal end of the membrane of the gB molecule. According to the principle of three-dimensional simulation and maintaining conformation of spherical domain, two novel candidate vaccine antigens, gB1 and gB2, were designed in this study. The fusion gene encoding gB1 and gB2 was amplified by recombinant PCR and cloned into mammalian cell expression vector pCI-neo. Transient expression of the protein was carried out by adherent 293FT cells. The target protein was purified by nickel affinity chromatography. Western blot analysis showed that the two recombinant proteins were antigenicity. Using CpG-ODN Al (OH) 3 as the adjuvant of gB1 and gB2, the high titer immune serum of two recombinant proteins was prepared by intramuscular injection of BALB/c mice in five weeks. The titers of antigen-specific IgG antibody detected by ELISA were 1: 6400, 1: 51200 and 1: 25600, 1: 51 200, respectively. The neutralization test of HCMV-VR1578 virus expressing GFP with immune serum was carried out. The results showed that the neutralization titers of gB1 and gB2 immunized serum were 1: 51.8 and 1: 24.1, respectively, in the absence of complement, the neutralization titers of gB1 and gB2 were 1: 51.8 and 1: 24.1, respectively. After adding rabbit complement, it was 1: 744 and 1: 264, respectively. In conclusion, two novel HCMV-gB candidate vaccine antigens were successfully designed and validated, which laid a foundation for further study of HCMV subunit vaccine.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2426855
[Abstract]:Human cytomegalovirus (Human cytomegalovirus,HCMV) belongs to 尾 subfamily of herpesvirus family. Vaccination may be an effective measure to prevent and control HCMV infection in some high-risk population. The genetic engineering vaccine based on virus surface glycoprotein (gB) has shown good protective effect in the second phase clinical trial of pregnant women and transplant patients. In order to further enhance the immune protection of gB vaccine, molecular design and immunological evaluation of novel gB vaccine antigen were carried out in this study. According to the published crystal structure of HSV-1-gB, the 3D structure of gB was obtained by on-line simulation of SWISS-Prot protein crystal database. The simulation results show that all three neutralizing epitopes, AD-1,AD-2 and DLD, are located in the spherical domain at the distal end of the membrane of the gB molecule. According to the principle of three-dimensional simulation and maintaining conformation of spherical domain, two novel candidate vaccine antigens, gB1 and gB2, were designed in this study. The fusion gene encoding gB1 and gB2 was amplified by recombinant PCR and cloned into mammalian cell expression vector pCI-neo. Transient expression of the protein was carried out by adherent 293FT cells. The target protein was purified by nickel affinity chromatography. Western blot analysis showed that the two recombinant proteins were antigenicity. Using CpG-ODN Al (OH) 3 as the adjuvant of gB1 and gB2, the high titer immune serum of two recombinant proteins was prepared by intramuscular injection of BALB/c mice in five weeks. The titers of antigen-specific IgG antibody detected by ELISA were 1: 6400, 1: 51200 and 1: 25600, 1: 51 200, respectively. The neutralization test of HCMV-VR1578 virus expressing GFP with immune serum was carried out. The results showed that the neutralization titers of gB1 and gB2 immunized serum were 1: 51.8 and 1: 24.1, respectively, in the absence of complement, the neutralization titers of gB1 and gB2 were 1: 51.8 and 1: 24.1, respectively. After adding rabbit complement, it was 1: 744 and 1: 264, respectively. In conclusion, two novel HCMV-gB candidate vaccine antigens were successfully designed and validated, which laid a foundation for further study of HCMV subunit vaccine.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
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1 罗丹;方峰;;人巨细胞病毒疫苗的相关研究进展[J];传染病信息;2006年03期
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