狂犬病减毒脂质体口服冻干活疫苗免疫效果检测

将制备的狂犬病rSRV9减毒口服冻干脂质体活疫苗配合以自制的复合佐剂,免疫小鼠、犬、猫,根据ELISA抗体定量检测方法,对免疫后小鼠血清IgG、小鼠粪便IgA、犬和猫血清IgG、犬和猫唾液IgA的ELISA抗体水平进行检测,其检测结果与注射狂犬病疫苗免疫效果进行对比。结果显示,复合佐剂+rSRV9病毒口服免疫后70d,小鼠血清抗狂犬病特异性IgG抗体水平为4 214.00U/mL,小鼠粪便中SIgA抗体水平为137.00U/mL;复合佐剂+rSRV9病毒口服免疫犬120d抗体水平最高,犬抗狂犬病特异性IgG抗体水平为1 420.00U/mL,唾液中SIgA抗体水平为1 199.00U/mL;猫抗狂犬病特异性IgG抗体水平为2 088.00U/mL,唾液中SIgA抗体水平为1 896.00U/mL。狂犬病疫苗口服组与注射组特异性抗体水平差异均不显著。结果表明,狂犬病rSRV9减毒口服冻干脂质体活疫苗的抗体产生水平已接近注射狂犬病疫苗的免疫效果,能够达到对动物的免疫效果。     

CDV-N重组狂犬病病毒的毒力及其免疫原性测定

为了探究CDV-N重组狂犬病病毒的毒力及对小鼠的免疫效果,将CDV-N重组狂犬病病毒于BSR细胞上扩毒培养、浓缩后测定其FFD₅₀及LD₅₀;将浓缩后的重组病毒液与复合佐剂按一定比例混匀,按不同免疫剂量、不同免疫方式分组免疫接种小鼠,利用ELISA试剂盒对狂犬病、犬瘟热的特异性抗体进行定性/定量检测。结果显示,重组病毒浓缩后的FFD₅₀值为7.85logFFD₅₀/0.1 mL,LD₅₀值为7.67logLD₅₀/0.03mL;免疫接种后第14天抗体阳性率达100%,小鼠血清中的特异性抗体IgG水平随着免疫剂量的递增而递增。试验结果可为CDV-N重组狂犬病病毒制备口服弱毒疫苗的进一步研究提供试验数据。     

狂犬病口服疫苗SRV9毒株基因序列的生物信息学分析

为了解狂犬病口服疫苗SRV₉毒株的基因序列与生物学特性的关系,并就其主要抗原位点与国内外现用狂犬病疫苗生产毒株进行比较,明确其作为口服疫苗株的分子基础,本试验通过RT-PCR方法对狂犬病口服疫苗SRV₉毒株的N、P、M、G、L基因分别进行克隆,并分别连接于pMD19-T载体,经酶切、测序鉴定后,用DNAStar软件对全基因序列进行分析,并与11株目前生产用狂犬病疫苗株进行基因序列比对分析和主要抗原位点的比较.狂犬病口服疫苗SRV₉毒株N、P、M、G、L基因序列与其他毒株的序列同源性为81.8%~100.0%,由全基因组进化树可知,SRV₉毒株与所有疫苗株在同一个大的分支内,均属于基因Ⅰ型,其中狂犬病口服疫苗SRV₉毒株与ERA、SAD B19、SAG-2等口服疫苗株的进化距离最近,而与疫苗生产毒株aG、RC-HL 之间的进化距离则较远.狂犬病口服疫苗SRV₉毒株与各疫苗株的糖蛋白不同结构区的氨基酸同源性分析表明,狂犬病口服疫苗SRV₉毒株与SAD B19、SAG-2、ERA口服疫苗株的膜外区、跨膜区和膜内区的同源性最高.狂犬病口服疫苗SRV₉毒株的基因组结构和抗原基因位点特征均与目前生产用口服疫苗株相似,这为今后狂犬病口服疫苗的研制和通过分子生物学技术构建基因重组弱毒疫苗株的研究提供了试验依据.     

基因重排狂犬病病毒疫苗株免疫效果的初步研究

试验探究了基因重排狂犬病病毒(RV)弱毒疫苗株能否刺激小鼠产生RV抗体免疫球蛋白G(IgG),比较了其不同免疫方式、不同免疫剂量对小鼠抗体水平的影响及安全性评估。选取28只体重(20±3)g、35日龄左右的昆明系小白鼠,随机分为7组:第1组为低剂量口服组:复合佐剂50μL+病毒液50μL;第2组为中等剂量口服组:复合佐剂150μL+病毒液150μL;第3组为高剂量口服组:复合佐剂300μL+病毒液300μL;第4组为低剂量注射组:复合佐剂50μL+病毒液50μL;第5组为中等剂量注射组:复合佐剂150μL+病毒液150μL;第6组为高剂量注射组:复合佐剂300μL+病毒液300μL;第7组为口服对照组:复合佐剂300μL+SRV9亲本毒株300μL,分别于0、7、14d进行免疫,采用ELISA定量检测试剂盒对各免疫组小鼠的血清IgG水平进行检测,免疫期结束后取各免疫组小鼠的肝脏、肾脏、肺脏、脑组织制作病理组织切片对疫苗的安全性进行评估。结果显示,各免疫组均产生了RV抗体IgG;肌内注射免疫产生的抗体速度较口服免疫快,但肌内注射免疫会导致小鼠发病死亡;600μL口服免疫组产生的抗体水平显著高于100、300μL口服免疫组及亲本毒株SRV9对照组(P<0.05),说明适当加大口服免疫剂量会使口服免疫组产生的抗体水平增加,并且不会引起不良反应。病理组织学检查发现,各口服免疫组小鼠的肝脏、肾脏、肺脏、脑组织的形态结构较为正常,未发生病变,表明基因重排RV疫苗株口服免疫副作用小、安全性较好。本试验结果为研制新型口服疫苗开辟了新的方向。     

基因重排狂犬病病毒疫苗株免疫效果的初步研究

试验探究了基因重排狂犬病病毒(RV)弱毒疫苗株能否刺激小鼠产生RV抗体免疫球蛋白G(IgG),比较了其不同免疫方式、不同免疫剂量对小鼠抗体水平的影响及安全性评估。选取28只体重(20±3)g、35日龄左右的昆明系小白鼠,随机分为7组:第1组为低剂量口服组:复合佐剂50μL+病毒液50μL;第2组为中等剂量口服组:复合佐剂150μL+病毒液150μL;第3组为高剂量口服组:复合佐剂300μL+病毒液300μL;第4组为低剂量注射组:复合佐剂50μL+病毒液50μL;第5组为中等剂量注射组:复合佐剂150μL+病毒液150μL;第6组为高剂量注射组:复合佐剂300μL+病毒液300μL;第7组为口服对照组:复合佐剂300μL+SRV9亲本毒株300μL,分别于0、7、14d进行免疫,采用ELISA定量检测试剂盒对各免疫组小鼠的血清IgG水平进行检测,免疫期结束后取各免疫组小鼠的肝脏、肾脏、肺脏、脑组织制作病理组织切片对疫苗的安全性进行评估。结果显示,各免疫组均产生了RV抗体IgG;肌内注射免疫产生的抗体速度较口服免疫快,但肌内注射免疫会导致小鼠发病死亡;600μL口服免疫组产生的抗体水平显著高于100、300μL口服免疫组及亲本毒株SRV9对照组(P0.05),说明适当加大口服免疫剂量会使口服免疫组产生的抗体水平增加,并且不会引起不良反应。病理组织学检查发现,各口服免疫组小鼠的肝脏、肾脏、肺脏、脑组织的形态结构较为正常,未发生病变,表明基因重排RV疫苗株口服免疫副作用小、安全性较好。本试验结果为研制新型口服疫苗开辟了新的方向。     

虎源FPV主要抗原表位区基因的原核表达及免疫原性分析

为筛选出制备猫瘟热亚单位疫苗的最佳佐剂类型与首选抗原片段,本试验对虎源FPV-HLJ株VP2全长基因及其截短基因片段PAB进行原核表达,切胶法纯化的表达蛋白经Western blot分析后,分别与氢氧化铝胶佐剂及弗氏佐剂混合,免疫BALB/c小鼠,间接ELISA方法检测小鼠体液免疫水平,初步评价各疫苗的免疫效果。结果表明:VP2、PAB融合蛋白主要以包涵体形式表达,切胶法获得的高纯度表达蛋白均能与鼠抗虎源FPV阳性血清发生特异性反应。纯化蛋白各佐剂免疫组免疫小鼠后均引起较强的特异性抗体IgG反应,其中PAB蛋白各免疫佐剂组抗体水平明显高于VP2蛋白相应免疫佐剂组,且PAB蛋白+氢氧化铝胶组与PAB蛋白+弗氏佐剂组抗体水平无显著差异(P0.05),但与未加佐剂组差异显著(P0.01)。研究证实,PAB蛋白具有良好的免疫原性,可与氢氧化铝胶佐剂协同用于FPV亚单位疫苗的制备。     

人参茎叶皂甙对口蹄疫病毒灭活疫苗免疫后抗体产生的影响

分别用人参茎叶皂甙(GSL)、白油或两者混合物作为免疫佐剂,与Asia-Ⅰ型口蹄疫病毒(FMDV)灭活疫苗混合免疫小鼠,检测免疫后的血清特异性抗体水平.结果表明,GSL具有佐剂作用,能显著促进机体产生抗FMDV抗体的水平,其佐剂作用呈量效关系.当GSL和白油混合形成复合佐剂时,具有组合效应,能产生比2种佐剂单独应用时更强的抗体效价;且GSL和白油混合产生的组合效应主要促进IgG2a和IgG2b的产生.     

5种佐剂对猪圆环病毒2型灭活疫苗免疫效果的影响

为了筛选得到可以显著提高猪圆环病毒2型(PCV2)灭活疫苗免疫效果的佐剂,本研究将ISA 206、ISA 201和市场上常见的3种其他佐剂分别与抗原进行乳化,制备成疫苗后进行小鼠免疫,同时设置空白对照组和商品疫苗对照组,测定小鼠血清中ELISA抗体的水平。结果显示佐剂2、3、5产生的抗体水平显著超过其他试验组,同时抗原稳定剂与佐剂3和佐剂5复配使用,抗体水平显著提升。试验结果的得出可以为开发新型高效的圆环病毒疫苗提供一定的数据支持。     

猪血凝性脑脊髓炎病毒不同佐剂灭活疫苗免疫BALB/c小鼠的比较试验

用血凝/血凝抑制试验（HA/HI）检测自制的猪血凝性脑脊髓炎病毒（HEV）氢氧化铝胶灭活苗和蜂胶佐剂灭活苗免疫BALB/c小鼠后的抗体效价,依据小鼠抗体效价比较不同免疫佐剂的效果。结果表明,用氢氧化铝胶佐剂配制的疫苗可刺激接种动物产生快速免疫应答反应,抗体产生效价高、维持时间长,其效果优于其它佐剂,有望成为HEV灭活疫苗的候选佐剂。     

PRRSV GP5和M蛋白滴鼻免疫小鼠后特异性抗体的中和活性分析

为探讨猪繁殖与呼吸综合征病毒(PRRSV)GP5和M蛋白滴鼻免疫小鼠后产生的特异性抗体的中和活性,研究以原核表达的重组蛋白GP5和M为抗原分别与霍乱毒素B亚基(CTB)佐剂按10∶1的比例混合制备成CTB黏膜佐剂疫苗,滴鼻免疫小鼠,于首次免疫后0,7,14,21,28,35,42 d收集唾液和血清样品,利用间接ELISA方法分别检测免疫小鼠唾液中特异性抗体IgA,结果均于28 d时S/N值达到最高,ELISA效价均为1∶2;血清中特异性抗体IgG分别于21,28 d时S/N值达到最高,ELISA效价均为1∶800。通过本实验室建立的基于PAM细胞中和试验方法分别检测唾液中特异性抗体IgA和血清中特异性抗体IgG中和滴度均1∶2,结果表明PRRSV GP5和M蛋白滴鼻免疫小鼠后产生的特异性抗体未达到免疫保护作用的中和抗体水平。     

Construction of Full-length cDNA of Recombinant Rabies Virus SRV9 Strain Expressing EgM123 Gene of Echinococcus granulosus

The aim of the experiment was to construct the recombinant rabies virus SRV₉ vaccine strain with EgM123 gene by reverse genetics technology and provide the technical means for effective prevention and control of rabies and hydatidosis in China's agricultural and pastoral areas.In this study,the structural protein N,P and L genes of rabies virus SRV₉ were synthesized using gene synthesis technology,which was based on the complete genome sequence of rabies virus SRV₉ and the fusion fragment of the N-P-M fusion fragment and the rabies G gene,through the carrier of enzyme insertion connection methods,the recombinant rabies virus L gene,N-P-M gene fusion fragment and G+EgM123+eGFP gene fusion fragment were successively recombined on the expression vector pcDNA3.1(-) to construct the full-length cDNA of recombinant rabies virus SRV₉ with EgM123 gene.The synthesized genes were constructed on pcDNA3.1(-) expression vector,and the results of transformation,plasmid digestion and gene sequencing showed that the length of N,P,L,N+P+M and G+EgM123+eGFP gene fragments were 1 365,1 107,6 471,3 160 and 3 256 bp,respectively.The full-length cDNA fragment of EgM123 gene recombinant rabies virus full-length cDNA was 12 465 bp,and the sequencing results of each gene fragment were 100%.In this experiment,the full-length cDNA fragment of recombinant EgM123 rabies and eukaryotic expression vectors of the N,P and L genes of rabies virus were successfully constructed,which could save EgM123 gene recombinant rabies by reverse genetics,it also provided the reference for the development of rabies and hydatid disease combined gene recombinant oral live vaccine.     

细粒棘球绦虫EgM123基因重组狂犬病病毒SRV9株基因组全长cDNA的构建

试验旨在通过反向遗传学技术构建EgM123基因重组狂犬病病毒SRV₉疫苗株,为中国农牧区的狂犬病和包虫病的有效防控提供技术手段。本试验参照狂犬病病毒SRV₉株全基因组序列,利用基因合成技术分别合成狂犬病病毒的结构蛋白N、P、L基因,以及N-P-M基因融合片段和狂犬病病毒G基因、细粒棘球绦虫EgM123基因与增强型绿色荧光蛋白eGFP融合片段基因,通过载体酶切插入连接方法,依次将狂犬病病毒L基因、N-P-M基因融合片段和G+EgM123+eGFP基因融合片段重组于pcDNA3.1（-）表达载体上,构建EgM123基因重组狂犬病病毒SRV₉全长cDNA。将合成的基因分别构建于pcDNA3.1（-）表达载体,经转化、质粒酶切、基因测序鉴定结果表明,狂犬病病毒N、P、L、N+P+M和G+EgM123+eGFP基因片段长度分别为1 365、1 107、6 471、3 160和3 256 bp;EgM123基因重组狂犬病病毒全长cDNA片段长度为12 465 bp,各基因片段测序结果为100%。本试验成功构建了EgM123基因重组狂犬病病毒全长cDNA片段和狂犬病病毒N、P、L基因的真核表达载体,为通过反向遗传学拯救EgM123基因重组狂犬病病毒及狂犬病和包虫病二联基因重组口服活疫苗的研制提供参考。     

Safety and immunogenicity of a vaccine bait containing ERA strain of attenuated rabies virus.

Ninety percent of foxes fed commercial ERA vaccine in a specially designed bait developed rabies serum neutralizing antibodies. The vaccine bait did not cause clinical signs of rabies when consumed by foxes, raccoons, skunks, dogs, cats, cattle and monkeys. When presented, in the laboratory, to wild rodents of the species Microtus, Mus musculus and Peromyscus, the vaccine baits caused vaccine-induced rabies only in Mus musculus. Laboratory mice of the CD-1 and CLL strain were susceptible to vaccine-induced rabies; however, studies showed that transmission of virus to other animals did not occur. These studies suggest that the vaccine bait described could be useful in a rabies control program in areas where foxes and wild dogs are the principal vectors.     

Seroconversion in captive African wild dogs (Lycaon pictus) following administration of a chicken head bait/SAG-2 oral rabies vaccine combination

This study determined the proportion of captive juvenile and adult African wild dogs (Lycaon pictus) that developed protective titres of rabies neutralising antibodies following ingestion of a chicken head bait/SAG-2 oral rabies vaccine combination. A single chicken head containing 1.8 ml of SAG-2 vaccine (10(8.0) TCID50/ml) in a plastic blister was fed to each of eight adult and three juvenile wild dogs. Bait ingestion resulted in a significant rise in serum neutralising antibody titres. Overall seroconversion rate was eight out of 11 (72.7%), and all the puppies and five out of eight (62.5%) adults showed potentially protective levels of antibodies on day 31. The mean post-vaccination neutralising antibody titre was within the range reported to be protective against challenge with virulent rabies virus in other species.     

Duration of immunity in foxes vaccinated orally with ERA vaccine in a bait.

Red foxes (Vulpes vulpes) vaccinated orally with the ERA strain of rabies vaccine in a bait were challenged after 83 mo. Ten of 11 foxes that had seroconverted following vaccination resisted challenge with a virulent rabies virus which produced clinical signs of rabies in 6 of 6 unvaccinated foxes. Five of 11 vaccinated animals retained titers of rabies virus neutralizing antibody throughout the period. Although 6 of 11 had no detectable antibody at the time of challenge, 5 of these 6 resisted challenge and had an anamnestic response, as indicated by elevated titers of antibody when measured at day 77 postchallenge. These results show that foxes can be immunized successfully with a single oral dose of ERA vaccine, probably with protection against a lethal rabies challenge, for at least 7 y.     

Serum antibody response to soluble extract of the third-larval stage of Ostertagia ostertagi in cattle

Serum IgG, IgM, and IgA antibody responses against L₃ antigens of Ostertagia ostertagi were monitored by enzyme-linked immunosorbent assay (ELISA) after one, two or multiple sequential inoculations of this nematode in calves. Following the first infection, antibody levels did not change. After a second inoculation, IgG increased significantly (P < 0.05) after 2 months. IgG was not significantly increased 1 month after challenge inoculation. IgM and IgA antibody levels did not change following the first or second inoculations of L₃. IgG antibody levels rose only slightly following multiple sequential inoculations with infectious L₃.

Results indicate that calves with ostertagiasis have very weak serum antibody responses to L₃, and these appear to be of little value in detection of the infection in these animals.     

Effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice

OBJECTIVE: To evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus. ANIMALS: 2,213 mice. PROCEDURE: Inactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus. RESULTS: Vaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.