肠道病毒71型VP1抗原表位多肽疫苗的实验研究

Experimental study of the epitope peptide vaccine against the structural protein VP1 of enterovirus 71 strain

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。

Objective To make the epitope peptide vaccine by using the structural protein VP1 of enterovirus 71( EV71) strain isolated from Guizhou area and investigate the immune protective effects on mice. Methods The VP1 amino acid sequences of 17 EV71 strains isolated from throat swab samples of children patients with hand-foot-mouth disease between March 1,2013 and December 31,2015 in Guizhou area were consistent( a total of 297 amino acids). Peptide fragments were designed and synthesized by using the amino acid sequence,that the last one was 27 amino acids,the others were 20 amino acids. The epitope peptide vaccine was made by using the polypeptide fragments with better immunogenicity,which was screened out by indirect ELISA method and Ig G positive serum of children who recovered from EV71 infection. Then the epitope peptide vaccine was used to protect healthy ICR female rats and the control group was set that female rats weren't inoculated( con). The neonatal rats were randomly divided into 2 subgroups of injected and non injected EV71 virus. The mice were killed 2 days after injection of the virus,then the tissue was harvested from the skeletal muscle,the small intestine and brain of all neonatal rats which was used to check EV71 virus by RT-PCR and agarose gel electrophoresis and observed the pathological lesion by H&E. Results 3 peptide vaccines were made by using polypeptide fragments with better immunogenicity screened from 19 segments polypeptide fragments with cross sequences. The female mice were immunized by using the peptide vaccines and the subsequent experiments were carried out in neonatal rats. RTPCR results showed that EV71 virus could be detected in infected groups,could not be detected in uninfected groups. The pathological lesion showed that skeletal muscle,intestine and brain tissue had obvious inflammatory changes in the neonatal rats infected with EV71 virus but the female mice weren't immunized. Inflammatory changes were significantly alleviated in the neonatal rats infected with EV71 virus and the female mice were immunized,there was no significant difference in each tissue between the three vaccinated groups. No obvious inflammatory lesion was found in each tissue of the neonatal rats which didn 't infected with EV71 virus and the female mice were immunized. Conclusion In our study,VP1 epitope peptide vaccine was successfully prepared,and its efficacy and safety were confirmed in animal experiments,it can provide methodological reference for the development of related vaccines in the future.