重组EV71、CA16型手足口病双价VLP疫苗的初步研究

发布时间：2018-11-14 18:33

【摘要】：目的运用Bac-to-Bac杆状病毒表达系统构建并表达重组EV71、CA16病毒样颗粒（Virus-like particle，VLP），对重组EV71、CA16双价VLP疫苗的免疫原性及免疫保护性进行评价，为发展安全、有效、具有保护作用的手足口病新型疫苗奠定基础。方法（1）通过测序分别获得构建EV71、CA16VLP所必需的疫苗候选株基因P1、3CD片段序列，首先根据昆虫密码子偏爱性选择优势密码子，运用分子生物学技术对EV71、CA16P1和3CD基因进行设计和全基因合成。将合成后的P1、3CD序列构建到穿梭质粒pFastBacDual上，通过位点特异性重组筛选阳性重组菌DH10BacTME.coli，将EV71、CA16的重组杆粒Bacmid转染昆虫细胞Sf9获得重组杆状病毒；通过透射电镜、间接免疫荧光、SDS-PAGE、Western blot对纯化的重组EV71、CA16VLP蛋白抗原进行鉴定。（2）将纯化后的重组目的蛋白，联合使用Al(OH)3佐剂，采用腹腔注射的途径免疫4-6周龄ICR小鼠，于0d、14d对小鼠进行两次免疫，一免10d和二免10d后采集小鼠尾静脉血，二免14d后取小鼠脾脏。通过间接ELISA法检测血清IgG抗体效价、血清抗体亚型分布及ELISPOT等方法比较重组EV71、CA16单价及双价VLP疫苗免疫原性；通过体外微量中和试验比较重组EV71、CA16单价及双价VLP疫苗对现有EV71、CA16流行株的保护作用；将EV71、CA16单价及双价VLP疫苗免疫ICR乳母鼠，用EV71、CA16强毒株攻击其新生5日龄乳鼠，观察乳鼠体重变化及存活率，，综合比较重组EV71、CA16单价及双价VLP疫苗的免疫保护效果。结果（1）经SDS-PAGE、Western blot以及间接免疫荧光试验结果均证明了EV71、CA16VLP的表达，透射电镜观察可见直径大小约为23～30nm的EV71、CA16VLP，与完整的病毒颗粒结构一致，通过蔗糖密度梯度离心纯化，获得了纯度在90%以上的EV71、CA16VLP抗原；（2）动物实验比较重组EV71、CA16VLP单价、双价疫苗的免疫原性。间接ELISA法显示重组EV71、CA16单价、双价VLP疫苗组与PBS对照组抗体水平差异显著（p0.05），且双价组显著高于单价组（p0.05）。中和抗体效价测定显示双价组中和抗体效价高于单价组（p0.05）。小鼠脾淋巴细胞中IFN-γ和IL-4分泌细胞水平检测显示重组EV71、CA16VLP疫苗各组均可以检测到一定水平的IFN-γ和相对较低水平的IL-4，且双价疫苗组高于单价组。应用EV71-BJ、CA16-TS强毒株分别攻毒，证实重组EV71、CA16单价、双价VLP疫苗均能有效保护5日龄ICR乳鼠抵抗致死剂量病毒的攻击，且双价VLP疫苗保护效果优于单价VLP疫苗。结论通过昆虫杆状病毒表达系统成功构建、表达和纯化EV71、CA16VLP，纯度可达90%以上。研制的重组V71、CA16双价VLP疫苗具有良好的免疫原性，能够刺激机体产生体液免疫应答和细胞免疫应答。昆虫杆状系统表达的重组EV71、CA16双价VLP疫苗具有较好免疫原性及免疫保护效果，是值得深入研究的手足口病候选疫苗。
[Abstract]:Objective to construct and express recombinant EV71,CA16 virus-like particles (Virus-like particle,VLP) by using Bac-to-Bac baculovirus expression system to evaluate the immunogenicity and immunogenicity of recombinant EV71,CA16 bivalent VLP vaccine. A new vaccine for HFMD with protective effect is established. Methods (1) the sequence of P1m3CD fragment of vaccine candidate gene necessary for EV71,CA16VLP construction was obtained by sequencing. Firstly, the dominant codon was selected according to insect codon preference, and EV71, was analyzed by molecular biology technique. CA16P1 and 3CD genes were designed and synthesized. The synthesized p1h3CD sequence was constructed into shuttle plasmid pFastBacDual, and the recombinant baculovirus was obtained by site-specific recombinant screening of positive recombinant strain DH10BacTME.coli, and transfection of the recombinant Bacmid of EV71,CA16 into insect cell Sf9. The purified recombinant EV71,CA16VLP protein antigen was identified by transmission electron microscopy indirect immunofluorescence and SDS-PAGE,Western blot. (2) the purified recombinant target protein was combined with Al (OH) 3 adjuvant. ICR mice aged 4-6 weeks were immunized by intraperitoneal injection. The mice were immunized twice on day 14. The caudal vein blood was collected after 10 days and 10 days, and the spleen was taken after 14 days. The immunogenicity of recombinant EV71,CA16 monovalent and bivalent VLP vaccine was compared by indirect ELISA assay, serum IgG antibody titer, distribution of serum antibody subtypes and ELISPOT. To compare the protective effect of recombinant EV71,CA16 monovalent and bivalent VLP vaccine on existing EV71,CA16 epidemic strains by in vitro microneutralization test. EV71,CA16 monovalent and bivalent VLP vaccines were used to immunize ICR lactating female mice. The neonatal 5-day-old mice were attacked with EV71,CA16 virulent strain to observe the body weight change and survival rate, and to compare the immune protective effect of recombinant EV71,CA16 monovalent and bivalent VLP vaccine. Results (1) the expression of EV71,CA16VLP was confirmed by SDS-PAGE,Western blot and indirect immunofluorescence assay. The structure of EV71,CA16VLP, with a diameter of about 23~30nm was the same as that of intact virus particles observed by transmission electron microscope. Purified by sucrose density gradient centrifugation, the EV71,CA16VLP antigen with purity above 90% was obtained. (2) the immunogenicity of recombinant EV71,CA16VLP monovalent and bivalent vaccine was compared in animal experiments. Indirect ELISA assay showed that there was significant difference in antibody level between the bivalent VLP vaccine group and the PBS control group (p0.05), and the level of antibody in the bivalent group was significantly higher than that in the univalent group (p0.05). Neutralization antibody titer test showed that neutralization antibody titer in bivalent group was higher than that in univalent group (p0.05). The levels of IFN- 纬 and IL-4 secreting cells in spleen lymphocytes of mice showed that the recombinant EV71,CA16VLP vaccine could detect a certain level of IFN- 纬 and a relatively low level of IL-4, in each group, and the bivalent vaccine group was higher than the univalent group. Using EV71-BJ,CA16-TS virulent strain to attack the virus separately, it was proved that the recombinant EV71,CA16 monovalent and bivalent VLP vaccine could effectively protect the 5-day-old ICR neonatal mice from lethal dose virus attack, and the bivalent VLP vaccine was better than the univalent VLP vaccine. Conclusion the purity of EV71,CA16VLP, expressed and purified by insect baculovirus expression system was over 90%. The recombinant V71 CA16 bivalent VLP vaccine has good immunogenicity and can stimulate humoral and cellular immune responses. The recombinant EV71,CA16 bivalent VLP vaccine expressed by insect rod system has good immunogenicity and immuno-protective effect. It is a candidate vaccine for HFMD.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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