肠道病毒71型VP1抗原表位多肽疫苗的实验研究

发布时间：2018-03-22 05:39

本文选题：手足口病　切入点：肠道病毒EV　出处：《中国循证儿科杂志》2017年05期 　论文类型：期刊论文

【摘要】：目的根据贵州省手足口病患儿肠道病毒71型(EV71)分离株的VP1结构蛋白序列研制多肽疫苗并研究其对小鼠的免疫保护作用。方法 2013年3月1日至2015年12月31日从贵州省手足口病患儿的咽拭子样本中分离到17株EV71毒株,其VP1氨基酸序列一致(共297个氨基酸),据此设计并合成多肽片段(除最后一段为27个氨基酸外,其余均为20个氨基酸);用感染EV71的手足口病病愈患儿的阳性血清,通过间接ELISA法筛选出免疫原性较好的多肽片段,制成多肽疫苗;用多肽疫苗免疫ICR健康雌鼠,并设未免疫的对照(Con);将雌鼠的子代小鼠随机分为注射和不注射EV71病毒颗粒的2个亚组;注射病毒2 d后处死所有小鼠,分别取小鼠的骨骼肌、小肠和脑组织,通过RT-PCR检测病毒感染情况,同时HE染色后在显微镜下观察病理改变。结果共设计并合成19段含交叉序列的多肽片段,从中筛选出3段免疫原性较好的(P2:VSRALTHALPAPTGQNTQVS;P4:ALQAAEIGASSNASDESMIE;P8:YANWDIDITGYAQMRRKVEL),制成多肽疫苗并免疫雌鼠,取雌鼠的子代小鼠进行后续试验。RT-PCR结果显示,各感染病毒亚组的骨骼肌、小肠和脑组织中均能检测到EV71病毒,而未感染病毒亚组均检测不到;病理结果显示,未接种多肽疫苗母鼠的子代小鼠在感染EV71病毒后,骨骼肌、小肠和脑组织均出现明显的炎性改变;接种多肽疫苗母鼠的子代小鼠骨骼肌、小肠和脑组织的炎性改变明显减轻,3种多肽疫苗对EV71所致炎症的改善作用无明显差异;接种多肽疫苗母鼠的未注射病毒亚组子代小鼠骨骼肌、小肠和脑组织中均未检测到EV71病毒,也未观察到明显的炎性改变。结论本研究成功制备VP1抗原表位多肽疫苗并在动物实验中证实了其有效性和安全性,可为今后相关疫苗的研制提供方法学借鉴。
[Abstract]:Objective to develop a polypeptide vaccine based on the VP1 structural protein sequence of enterovirus 71 EV71 isolated from children with hand-foot-mouth disease in Guizhou province and to study its immuno-protective effect on mice. Methods from March 1, 2013 to December 31, 2015, a polypeptide vaccine was prepared from Guigui. Seventeen strains of EV71 were isolated from throat swabs of children with hand, foot and mouth disease. Its VP1 amino acid sequence was identical (297 amino acids), according to which the peptide fragments were designed and synthesized (except for 27 amino acids in the last segment, the rest were 20 amino acids). Polypeptide vaccine was prepared by indirect ELISA method, and ICR healthy female mice were immunized with polypeptide vaccine. The offspring of female mice were randomly divided into two subgroups of injection and non-injection of EV71 virus particles. After 2 days of injection of the virus, all the mice were killed and the skeletal muscle, small intestine and brain tissue of the mice were taken, respectively. The virus infection was detected by RT-PCR, and the pathological changes were observed under microscope after HE staining. Results A total of 19 polypeptide fragments containing cross sequences were designed and synthesized. Three segments of VSRALTHALPAPTGQNTQQVSU P4ALQAAEIGASSNASDESMIEN P8: YANWDIDITGYAQRKVELL were selected from the three segments of immunogenicity to make polypeptide vaccine and immunize female mice. The results of follow up test .RT-PCR showed that EV71 virus could be detected in skeletal muscle, small intestine and brain tissue of each subgroup of infected mice. The pathological results showed that there were obvious inflammatory changes in skeletal muscle, small intestine and brain tissue after infection of EV71 virus in the offspring of mice without polypeptide vaccine. The inflammatory changes of small intestine and brain tissues in the skeletal muscle, small intestine and brain tissues of maternal mice inoculated with polypeptide vaccine had no significant difference in ameliorating the inflammation induced by EV71. No EV71 virus was detected in the skeletal muscle, small intestine and brain tissues of the uninjected virus subgroup of mice inoculated with polypeptide vaccine. Conclusion VP1 epitope peptide vaccine has been successfully prepared and proved to be effective and safe in animal experiments, which may provide methodological reference for the development of related vaccines in the future.
【作者单位】：遵义医学院研究生院;遵义医学院第三附属医院儿童重症医学科;
【基金】：国家自然科学基金项目:81260292 遵义市科技局科技计划基金项目:遵市科合社字【2013】14号遵义市科学技术基金项目遵市科合人才【2015】24号遵义市“15851人才精英工程”培养人才在研项目经费资助项目:2013-20
【分类号】：R392

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