【作者】 张爽；

【导师】 贾立军； 金宁一； 李昌；

【作者基本信息】 延边大学 ， 兽医学， 2022， 硕士

【摘要】 猪δ冠状病毒(Porcine delta corona virus,PDCoV)是2012年新发现的一种猪肠道致病性冠状病毒,可以感染各年龄段猪群,哺乳期仔猪最易感。临床症状表现为急性水样腹泻、呕吐、脱水等,具有较强的致病性,可造成哺乳仔猪死亡。2014年美国多地相继暴发PDCoV导致的腹泻疫情,随后几年在全球广泛传播和流行,给全球养猪业造成严重经济损失。我国2016年江西省某猪场PDCoV的阳性检出率更是高达33.1%,更为重要的是PDCoV常与猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)存在共感染,不但加重了对猪群的危害,更是给两种病毒的诊断及预防带来困难。同时PDCoV具有跨物种传播的能力和极其广泛的宿主嗜性,能够感染包括人在内的多种哺乳动物和鸟类,对食品安全和公共卫生存在潜在危害。但目前尚无商品化的疫苗和有效的治疗手段防控PDCoV。受同类型的新型冠状病毒(Severe acute respiratory syndrome coronavirus,SARS-Co V-2)受体结合区(receptor-binding domain,RBD)相关疫苗研究启发,本试验以PDCoV受体结合区蛋白为研究对象,利用哺乳动物细胞和昆虫动物细胞两种不同的表达系统表达PDCoV受体结合区蛋白,并制备了小鼠PDCoV受体结合区蛋白多克隆抗体,以多克隆抗体血清为基础建立了小鼠PDCoV受体结合区间接ELISA方法;同时以制备的两种系统表达的PDCoV受体结合区蛋白为基础,制备了PDCoV受体结合区亚单位疫苗,免疫小鼠和仔猪后对相关指标进行检测,对其免疫原性进行评价;同时进行了仔猪攻毒试验。利用哺乳动物细胞系统成功表达出可溶性PDCoV受体结合区蛋白(MPDR),经Strep-Tactin XT亲和层析纯化后蛋白纯度可达到98%以上,以此为抗原免疫小鼠后成功制备出反应原性良好的多克隆抗体血清,效价可达到1:102400以上。以小鼠多克隆血清为阳性样本建立小鼠间接ELISA方法,对蛋白包被浓度、血清稀释倍数及作用时间、酶标二抗稀释浓度和作用时间、显色时间等条件进行优化,对所建立方法的特异性、灵敏性、重复性等因素进行评价,最终确定了ELISA检测最优反应条件:抗原最佳包被浓度为10μg/m L;血清最佳作用条件为1:400孵育30 min;酶标二抗最佳作用条件为1:3000孵育30 min;底物最佳显色时间为15 min;确定评价特异性抗体水平的临界值为0.2169,该方法与SARS-Co V-2、TGEV、PEDV等多种阳性血清均无交叉反应,灵敏度在1:51200以上,批内变异系数为2.93%-3.84%,批间变异系数为2.41%-3.98%,成功建立了小鼠PDCoV受体结合区蛋白抗体间接ELISA方法,可以应用于小鼠PDCoV受体结合区蛋白抗体水平的评价和分析。利用昆虫细胞表达系统在胞内成功表达出了具有反应原性的PDCoV受体结合区蛋白(BPDR),超速离心进行BPDR蛋白浓缩。将纯化后的MPDR蛋白和浓缩后的BPDR蛋白与佐剂(铝盐+Cp G)分别混合,制备成MPDR-AC和BPDR-AC两种亚单位疫苗免疫小鼠,测定小鼠体重变化、特异性抗体水平及效价、中和抗体水平、细胞亚群和细胞因子等指标,综合评级两种亚单位疫苗的免疫原性。结果表明,免疫后,小鼠体重除SF9-AC对照组外其余各组31 d内的体重均呈上升趋势;MPDR-AC和BPDR-AC免疫组特异性抗体在的第31 d达到较高水平,效价均为1:51200;MPDR-AC和BPDR-AC,免疫组第31 d的平均中和抗体水平分别为1:107和1:56;T淋巴细胞增值实验中MPDR-AC免疫组SI值是PBS对照组的3.84倍(P<0.01);CD3~+CD4~+淋巴细胞亚群升高百分比MPDR-AC组存在显著差异(P<0.05);经PDCoV刺激后淋巴细胞IL-4细胞因子均有一定水平的上升,MPDR-AC组(P<0.01)、BPDR-AC组(P<0.05)。与PBS、AC、BSF9-AC对照组相比MPDR-AC、BPDR-AC免疫组均能引起一定水平的免疫应答,且MPDR-AC组相较于BPDR-AC组表现出更好的免疫效果,故选择免疫效果更好的MPDR蛋白进行仔猪免疫与攻毒试验研究。MPDR蛋白与佐剂(铝盐+分子佐剂+Cp G)混合制备亚单位疫苗,对免疫后仔猪的体重、体温进行监测,同时对仔猪每周的特异性抗体水平变化进行分析,对特异性抗体水平最高的血清进行中和抗体检测。结果表明,免疫后仔猪体重持续上升、体温无明显波动;特异性抗体水平整体呈现上升趋势,并在第31 d达到峰值;4头MPDR-ACF组仔猪在第31 d特异性抗体效价介于1:12800-1:102400之间;第31 d血清MPDR-ACF免疫组的中和抗体是ACF对照组的3倍,且存在显著性差异(P<0.05)。对二免后的仔猪进行攻毒,检测仔猪体重、体温及粪便病毒载量,结果显示,攻毒后两组仔猪体温稳定在正常范围内,MPDR-ACF免疫组体重整体呈上升趋势,ACF对照组有1头仔猪出现体重下降、腹泻症状,MPDR-ACF免疫组粪便病毒载量整体呈现较低水平,ACF对照组整体病毒载量超过MPDR-ACF免疫组,结果表明,MPDR亚单位疫苗对于仔猪感染PDCoV具有一定的保护效果。本研究制备的MPDR亚单位疫苗能够引起小鼠和仔猪较高水平的免疫应答,而且能够对仔猪提供一定的保护,为PDCoV受体结合区蛋白的研究和PDCoV新型疫苗研发奠定了基础。更多还原

【Abstract】 Porcine delta coronavirus(PDCoV)is a newly discovered porcine intestinal pathogenic coronavirus in 2012,which can infect pigs of all ages,especially in lactating piglets.The clinical symptoms are acute watery diarrhea,vomiting and dehydration,which have strong pathogenicity and can cause the death of suckling piglets.Diarrhea caused by PDCoV broke out in many places in the United States in 2014,which spread and spread widely around the world in the following years,causing serious economic losses to the global pig industry.The positive rate of PDCoV in a pig farm in Jiangxi Province in China in 2016 was as high as 33.1%.More importantly,PDCoV is often co-infected with porcine epidemic diarrhea virus(PEDV),which not only aggravates the harm to pigs,but also brings difficulties to the diagnosis and prevention of the two viruses.At the same time,PDCoV has the ability of cross-species transmission and a wide range of host tropism,can infect a variety of mammals and birds,including human beings,and has potential harm to food safety and public health.However,there is no commercial vaccine and effective treatment to prevent and control PDCoV.Inspired by the same type of vaccine related to the receptor binding domain(RBD)of Severe acute respiratory syndrome coronavirus(SARS-Co V-2),this study took the PDCoV receptor binding domain protein as the research object,expressed PDCoV receptor binding domain protein in mammalian cells and insect animal cells,and prepared polyclonal antibodies against PDCoV receptor binding domain protein in mice.An indirect ELISA method for mouse PDCoV receptor binding region was established based on polyclonal antibody serum.At the same time,based on the PDCoV receptor binding domain proteins expressed in two systems,the PDCoV receptor binding domain subunit vaccine was prepared.After immunizing mice and piglets,the related indexes were detected and its immunogenicity was evaluated.The soluble PDCoV receptor binding domain protein(MPDR)was successfully expressed in mammalian cell system.The purity of the protein purified by Strep-Tactin XT affinity chromatography was more than 98%.The polyclonal antibody serum with good reactivity was successfully prepared after immunizing mice with this antigen,and the titer was more than 102400.A mouse indirect ELISA method was established by using mouse polyclonal serum as positive samples.The conditions such as protein coating concentration,serum dilution multiple and action time,enzyme labeled second antibody dilution concentration and action time,color time were optimized,and the specificity,sensitivity and repeatability of the established method were evaluated.Finally,the optimal reaction condition of ELISA detection was determined:the optimal antigen coating concentration was 10μg/m L.The best condition of serum was incubated with 1:400 for 30min,the best condition of enzyme labelled second antibody was incubated with 13000 HV for 30min,and the best color developing time of substrate was 15min.The critical value for the evaluation of specific antibody level was0.2169.There was no cross reaction with SARS-Co V-2,TGEV,PEDV and other positive sera.The sensitivity,intra-assay coefficient of variation and inter-assay coefficient of variation were 2.93%,3.84%and 2.41%,respectively.The indirect ELISA method of mouse PDCoV receptor binding domain protein antibody was successfully established.It can be used to evaluate and analyze the antibody level of PDCoV receptor binding domain protein in mice.The PDCoV receptor binding domain protein(BPDR)with reactivity was successfully expressed in insect cell expression system,and BPDR protein was concentrated by ultracentrifugation.Purified MPDR protein and concentrated BPDR protein were mixed with adjuvant(aluminum salt+Cp G)to prepare MPDR-AC and BPDR-AC subunit vaccines to immunize mice.The changes of body weight,specific antibody level and titer,neutralizing antibody level,cell subsets and cytokines were measured,and the immunogenicity of the two subunit vaccines were evaluated comprehensively.The results showed that after immunization,the body weight of mice in all groups except SF9-AC control group showed an upward trend within 31 days,and the specific antibodies of MPDR-AC and BPDR-AC immunized groups reached a higher level on the 31st day,and the titers of specific antibodies in MPDR-AC and BPDR-AC immunized groups were both 51200 and BPDR-AC,and the average neutralizing antibody levels on the 31st day of immunization group were 1:107 and 1:56,respectively.In T lymphocyte proliferation test,the SI of MPDR-AC immunized group was 3.84 times higher than that of PBS control group,the percentage of CD3~+CD4~+lymphocyte subsets increased significantly in MPDR-AC group,and IL-4 cytokines increased to some extent in MPDR-AC group and BPDR-AC group after PDCoV stimulation.Compared with PBS,AC and BSF9-AC control groups,MPDR-AC and BPDR-AC immunization groups could induce a certain level of immune response,and MPDR-AC group showed better immune effect than BPDR-AC group,so MPDR protein with better immune effect was selected for piglet immunity and challenge test.The subunit vaccine was prepared by mixing MPDR protein with adjuvant(aluminum salt+molecular adjuvant+Cp G).The body weight and body temperature of immunized piglets were monitored.At the same time,the weekly changes of specific antibody level of piglets were analyzed,and the serum with the highest specific antibody level was detected.The results showed that the body weight of piglets increased continuously and the body temperature did not fluctuate significantly after immunization,and the level of specific antibody increased as a whole and reached the peak on the 31st day,and the specific antibody titer of 4 piglets in the MPDR-ACF group was between 12800 and 102400 on the 31st day,and the neutralizing antibody in the serum MPDR-ACF immunized group was 3 times higher than that in the ACF control group on the 31st day.The body weight,body temperature and fecal virus load of the piglets after second immunization were detected.The results showed that the body temperature of the two groups was stable in the normal range,the body weight of the MPDR-ACF immunized group increased as a whole,one piglet in the ACF control group showed symptoms of weight loss and diarrhea,the fecal viral load of the MPDR-ACF immunized group showed a low level,and the overall viral load of the ACF control group was higher than that of the MPDR-ACF immunized group.The results showed that MPDR subunit vaccine had a certain protective effect on piglets infected with PDCoV.The MPDR subunit vaccine prepared in this study can induce a high level of immune response in mice and piglets,and can provide some protection to piglets,which lays a foundation for the study of PDCoV receptor binding domain proteins and the development of new PDCoV vaccines.更多还原