迟缓爱德华菌菌蜕疫苗对罗非鱼注射和口服免疫的效果分析

通过腹腔注射、口服两种免疫途径探讨迟缓爱德华菌菌蜕疫苗对罗非鱼Oreochromis niloticus的免疫保护效果。将制备的迟缓爱德华菌菌蜕疫苗（ETG）和福尔马林灭活疫苗（FKC）采用腹腔注射、口服两种免疫途径免疫罗非鱼,分别于免疫后14d、21d和28d采集罗非鱼血清、头肾、脾脏,测定血清中抗体IgM水平,血清中酸性磷酸酶（ACP酶）、超氧化物歧化酶（SOD酶）活性及罗非鱼头肾和脾脏中白介素（IL-1）、肿瘤坏死因子（TNF）、干扰素（IFN）、Caspase3等细胞因子的相对表达量,并通过攻毒试验得到菌蜕疫苗、福尔马林灭活疫苗两种疫苗的相对免疫保护率。免疫组罗非鱼的血清抗体水平均极显著高于（P<0.01）对照组,ETG注射组抗体效价极显著高于（P<0.01）FKC口服组。免疫28d,免疫组SOD、ACP酶活力显著高于（P<0.05）对照组（Group E、F）;在头肾中,免疫组（Group A、B）TNF、IL-1和IFN的相对表达量显著高于（P<0.05）对照组（Group E、F）。在免疫保护试验中,所有免疫组的免疫保护率均显著高于（P<0.05）对照组,注射、口服菌蜕疫苗的相对保护率分别为79%、77%,注射、口服灭活疫苗组的的相对保护率分别为62%、60%,但两种疫苗免疫保护率无显著差异。试验研制的菌蜕疫苗得到更高的免疫保护率,菌蜕疫苗在预防罗非鱼爱德华菌病中有良好的研究价值和应用前景。     

磁小体对小鼠免疫应答的影响

【目的】研究细菌磁小体对小鼠免疫应答的影响,为磁小体的体内应用提供参考依据。【方法】以卵清白蛋白(OVA)作为抗原分别与弗氏完全佐剂(阳性对照)、磁小体(BMP)悬液、PBS缓冲液(阴性对照)混合免疫小鼠。两周后检测血清中抗OVA特异性抗体及亚型、细胞因子表达水平以及淋巴细胞增殖能力。【结果】与阴性对照(PBS组)相比,BMP组在IL-2、IFN-γ和IL-4、IL-10的表达水平、淋巴细胞增殖能力、特异性抗体的产生没有明显差异。【结论】磁小体对小鼠免疫应答无显著的影响。     

三种创伤弧菌免疫原的制备及其对黄姑鱼的免疫保护效果

从创伤弧菌(Vibrio vulnificus)中提取菌体脂多糖(lipopolysaccaride,LPS)和外膜蛋白(outer membrane protein,OMP)并制备福尔马林灭活全菌苗(FKC),腹腔注射接种黄姑鱼。分别在注射第0、7、14、21和28天后测定了受免鱼血清中凝集抗体效价、血清溶菌酶活性和血液白细胞吞噬活性,以及免疫28d后的相对免疫保护率。结果表明,3种抗原对黄姑鱼均有较强的免疫原性。免疫后,免疫组血清凝集抗体效价逐渐增高,第28天时最高;溶菌酶活性(LMZ)、白细胞吞噬活性(PP)和吞噬指数(PI)显著升高(P<0.01),第21天达到峰值,随后逐渐下降。各组之间比较表明,受免后7、14、21和28d,免疫组黄姑鱼凝集抗体效价、PP、PI和LMZ显著高于对照组(P<0.05),LPS和OMP组凝集抗体效价低于FKC组,LPS和OMP组的相对免疫保护率高于FKC组,各组间免疫保护率大小顺序为LPS组>OMP组>FKC组>对照组。     

表达ETEC F41重组干酪乳杆菌口服及滴鼻免疫效果的比较

摘要：【目的】比较小鼠滴鼻与口服接种ETEC F41重组干酪乳杆菌后,机体所产生的抗ETEC F41的黏膜免疫和系统免疫及免疫保护率的差异,为确定ETEC乳酸菌疫苗免疫程序奠定基础。【方法】将构建的重组质粒pLA-F41电转化入干酪乳杆菌,获得阳性重组菌。重组菌在MRS 培养基中进行表达,经Western blot检测目的蛋白的表达,间接免疫荧光及流式细胞术检测外源蛋白展示到菌体表面。SPF级BALB/c小鼠随机分成4组,每组40只,将重组菌以滴鼻和口服途径分别接种2组小鼠,对照组分别接种同剂量的空质粒菌     

猪丹毒丝菌天然SpaA和重组SpaA-N免疫保护效果的评价

【目的】本试验以小鼠为动物模型,评估了猪丹毒丝菌重组表面保护性抗原A的N端保护区域(rSpaA-N)和天然SpaA的免疫保护效果。【方法】将猪丹毒丝菌C43311株SpaA-N以可溶形式表达在大肠杆菌BL21中,用GST Bind Resin纯化试剂盒纯化rSpaA-N,采用电洗脱法从猪丹毒丝菌C43311株NaOH提取抗原中纯化天然SpaA,将rSpaA-N、天然SpaA和NaOH提取抗原制成亚单位疫苗,同时设GST及生理盐水对照组,间隔2周分3次皮下免疫小鼠,第3次免疫后2周用100LD50猪丹毒丝菌C43065株进行腹腔攻毒,采用间接ELISA方法检测免疫组小鼠血清的抗体动态变化。【结果】SDS-PAGE结果显示,采用GST Bind Resin纯化试剂盒和电洗脱法纯化得到了66kDa的rSpaA-N和64kDa的天然SpaA,蛋白含量分别为1.34mg/mL和1.26mg/mL,而Western印迹结果表明rSpaA-N和纯化前后的SpaA具有良好的免疫反应性。保护试验结果表明,不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组均能完全保护小鼠受强毒株C43065的致死性攻击,而GST组和生理盐水组小鼠攻毒后全部死亡。ELISA检测结果表明,在不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组小鼠血清中的抗体效价之间无显著差异(P0.05)。【结论】本研究结果表明rSpaA-N具有良好的免疫保护作用,可以作为猪丹毒亚单位疫苗。     

嗜水气单胞菌菌蜕的制备及其对银鲫的口服免疫

菌蜕系统是一个自身具有佐剂性质的新型疫苗体系,不含细胞质内容物但具有细菌的完整表面抗原结构,可诱导机体的体液、细胞免疫应答及增强黏膜免疫反应.本研究通过将带有裂解基因E的质粒pElysis转化至嗜水气单胞菌J-1株中,对Ah J-1(pElysis)进行温度诱导,温度从28℃升至42℃,每隔15min检测菌液的OD600值,测定其溶菌动力学,并做无菌检验,用扫描电镜观察裂解后的细菌形态,研究其作为口服疫苗对银鲫的效果.结果显示,通过温度诱导,嗜水气单胞菌J-1(pElysis)OD值在诱导30min后开始持续下降,75min时开始趋于平稳,到120min溶菌效率达99.99%,诱导16h后进行无菌检验,证实其无活菌.扫描电镜观察绝大部分菌体经诱导后形成菌蜕,细胞两端有溶菌通道.动物试验表明,用菌蜕口服免疫的银鲫,在第5周产生较高的凝集抗体,达到27,并能维持2周;而甲醛灭活苗组为26,维持时间仅一周;生理盐水对照组效价仅2.攻击试验表明,菌蜕疫苗组和甲醛灭活疫苗组对嗜水气单胞菌强毒株J.1的攻击均有保护作用,其相对保护率分别为16/20(78.95%)和12/20(57.9%),显示菌蜕疫苗比普通灭活疫苗能更有效地激活机体的免疫保护.     

重组大肠杆菌菌蜕与幽门螺杆菌蛋白质疫苗协同免疫BALB/c小鼠

目的:考察重组菌蜕和蛋白质疫苗混合后对BAB/c小鼠机体免疫反应的影响。方法:首先构建展示尿素酶B抗原表位的大肠杆菌菌蜕,同时通过融合PCR构建分子内佐剂蛋白质疫苗rLTBKAT,然后分别利用该菌蜕和蛋白质疫苗免疫BAB/c小鼠。结果:当rLTBKAT和重组菌蜕联合口服免疫BAB/c小鼠时,其抗尿素酶B抗体的效价由单独免疫时的1∶(239±23)提高到1∶(681±76),二者差异极显著(P=0.009);而其抗过氧化氢酶抗体的效价则由单独免疫时的1∶(2800±275)下降至1∶(1800±400),二者差异不显著(P=0.08)。抗体分型试验表明,单独和联合免疫时,其抗尿素酶B抗体类型均以IgG1为主,而抗过氧化氢酶抗体类型则由单独免疫时的IgG1为主转变为联合免疫时的IgG2a为主。联合免疫时,小鼠抗尿素酶B和抗过氧化氢酶IgA抗体水平均高于单独免疫时。结论:重组菌蜕和蛋白质疫苗联合免疫,可提高机体对某些抗原的免疫水平或改变其反应类型。     

玉屏风散对鱼类非特异性免疫和免疫相关基因表达的影响

为探究传统中药玉屏风散在水产养殖上的生物学功效,在基础饲料中分别添加2.5%、5%和10%的玉屏风散和1%的芪参散配成实验饲料,饲喂实验鱼2周,然后分别观察两种中药对罗非鱼(体重约220 g)的生长、非特异性免疫及免疫相关基因表达的影响以及对草鱼(体重约20 g)免疫保护率的影响。结果显示:各中药组均提高了罗非鱼增重率, 10%玉屏风散组效果最显著;各实验组中罗非鱼肝、肾、脾体指数和各血液学指标与对照组无显著差异;各中药组罗非鱼谷草转氨酶(AST)和谷丙转氨酶(ALT)含量均不高于空白对照组; 2.5%、10%玉屏风散组和1%芪参散组碱性磷酸酶(AKP)含量均高于对照组(P0.05);各药物组罗非鱼溶菌酶活性均高于对照组,且2.5%玉屏风散组与对照组差异显著(P0.05);玉屏风散各剂量组罗非鱼呼吸暴发活性均高于对照组(P0.05);罗非鱼肝脏中热休克蛋白70(HSP70)在各实验组中表达量均上调,肾脏中HSP70在2.5%和5%玉屏风散组表达量上调,脾脏中HSP70在各实验组中表达量均下调;肝脏中转化生长因子受体TGFRⅢ在2.5%玉屏风散组表达量上调,肾脏中TGFRⅢ在2.5%玉屏风散组和1%芪参散组表达量上调,脾脏中TGFRⅢ在各实验组中表达量均上调;玉屏风散各剂量组能够提高草鱼对嗜水气单胞菌的抵抗能力,表现为死亡率降低和进入血液的细菌减少,其中5%玉屏风散组效果最佳。实验表明,玉屏风散能够有效促进罗非鱼生长,对其非特异性免疫指标以及免疫相关基因表达均有不同程度的促进作用,并能够有效地提高草鱼的免疫保护率。       

利用展示尿素酶B表位的大肠杆菌菌蜕递送幽门螺杆菌核酸疫苗

探讨和分析了重组大肠杆菌菌蜕递送系统能否进一步提高幽门螺杆菌核酸疫苗的免疫应答水平.首先构建了展示尿素酶B表位的重组大肠杆菌菌蜕,然后以此菌蜕包裹幽门螺杆菌核酸疫苗pcDNAKAT,经DNA凝胶电泳、荧光显微镜观察和FACS分析,表明该质粒DNA可以非特异地结合到大肠杆菌菌蜕内膜上.该核酸疫苗经重组菌蜕包裹后免疫小鼠,其血清抗过氧化氢酶抗体效价由单独免疫时的1∶(307±39)提高到1∶(520±54),二者差异极显著(P=0.005 8),结果表明利用菌蜕递送系统能较好地提高核酸疫苗的免疫应答水平.     

沙眼衣原体分泌性蛋白Pgp3滴鼻免疫增强小鼠对衣原体生殖道感染的保护作用

【目的】探讨不同免疫途径沙眼衣原体(Chlamydia trachomatis,Ct)分泌性蛋白Pgp3的免疫保护效果,分析其可能的保护机制,以确定Pgp3蛋白疫苗在Ct疫苗研制中的应用价值。【方法】分泌性蛋白Pgp3经滴鼻或肌注途径免疫雌性Balb/c小鼠,免疫60 d后,阴道接种鼠沙眼衣原体(Chlamydia muridarum,Cm)建立生殖道感染动物模型,在该模型中评价Pgp3蛋白疫苗抗Cm感染的保护效果,并探讨其机制。【结果】滴鼻或肌注免疫后,小鼠血清及生殖道中检测到了特异性抗体;小鼠脾淋巴细胞产生IFN-γ、IL-17及IL-5水平均明显高于对照组,且滴鼻免疫组IFN-γ水平升高较肌注组更显著(P<0.05);Pgp3蛋白滴鼻免疫组小鼠经Cm生殖道感染后,阴道带菌时间明显缩短,输卵管病理改变轻而肌注免疫组其保护作用不明显。【结论】Pgp3蛋白经滴鼻免疫可有效诱导小鼠产生明显的抗Cm保护效应。其可能的免疫保护机制与诱导Th1型为主的细胞免疫应答及高效价的特异性抗体有关,提示Pgp3蛋白疫苗具有潜在的疫苗研究与开发价值。     

Effect of oral immunization with Aeromonas hydrophila ghosts on protection against experimental fish infection

Aims:  To investigate whether oral immunization with Aeromonas hydrophila ghosts (AHG) vaccine can elicit mucosal and systemic immune responses of Carp ( Carassius auratus gibelio ) compared to conventional formalin-killed bacteria (FKC).
Methods and Results:  Fish were fed diets coated with AHG, FKC or phosphate buffered saline (PBS) alone, after immunization, more antigen-specific antibody was significantly detected in serum and intestinal mucus in AHG group than FKC group and PBS group. In addition, after challenged with the parent strain J-1, the survival of bacterial ghost-vaccinated fish was higher than PBS group and FKC group, the relative per cent survival (RPS) being 76·8%, 58·9%, respectively.
Conclusions:  Oral immunization with A. hydrophila ghosts can elicit systemic and mucosal adaptive immune responses and has higher potential to induce protective adaptive immunity than normal vaccine.
Significance and Impact of the Study:  Oral immunization with bacterial ghosts is a promising new solution with potential application to prevent diseases in fish.     

Immune responses and immune-related gene expression profile in orange-spotted grouper after immunization with Cryptocaryon irritans vaccine

In order to elucidate the immune-protective mechanisms of inactivated Cryptocaryon irritans vaccine, different doses of C. irritans theronts were used to immunize orange-spotted grouper (Epinephelus coioides). We measured serum immobilization titer, blood leukocyte respiratory burst activity, serum alternative complement activity, and serum lysozyme activity weekly. In addition, the expression levels of immune-related genes such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), major histocompatibility complexes I and II (MHC I and II), and transforming growth factor-β1 (TGF-β1) were determined in spleen and gills. The results showed that the immobilization titer, respiratory burst activity, and alternative complement activity of immunized fish were significantly increased, and the levels of the last two immune parameters in the high-dose vaccine group were significantly higher than in the low-dose vaccine group. Serum lysozyme activity in the high-dose vaccine group was significantly higher than in the PBS control group. Vaccination also regulated host immune-related gene expression. For example, at 2- and 3- weeks post immunization, IL-1β expression in the high-dose vaccine group spleen was significantly increased. At 4-weeks post immunization, the fish were challenged with a lethal dose of parasite, and the survival rates of high-dose vaccine group, low-dose vaccine group, PBS control group, and adjuvant control group were 80%, 40%, 0%, and 10% respectively. These results demonstrate that inactivated C. irritans vaccination improves specific and nonspecific immune responses in fish, enhancing their anti-parasite ability. These effects are vaccine antigen dose-dependent.     

重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

摘要：【目的】为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响。【方法】我们将HBV DNA疫苗（pcDS2）和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTT和流式细胞仪检测T细胞增殖反应,及流式细胞仪检测细胞因子表达水平。【结果】pcDS2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后, 联合免疫组刺激指数(SI)明显降低;经流式细胞仪检测进一步证实联合免疫组T细胞增殖反应被显著抑制;联合免疫组T细胞表达IL-10和Foxp3水平显著升高。【结论】pcDS2和HBsAg联合免疫能诱导产生特异性体液免疫应答,但不能诱导产生抗原特异性T细胞增殖反应;T细胞增殖反应被显著抑制可能与T细胞表达IL-10和Foxp3上调有关;本研究为急性乙型肝炎和爆发性乙型肝炎治疗及HBV疫苗的研究奠定了基础。     

Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.     

人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫活性

为了研制人乳头瘤病毒16型(HPV16)防治性疫苗,分析了HPV16 E5与IL-12联合基因疫苗的免疫活性。将构建的pcDNA3.1(+)/E5与pcDNA3.1(+)/IL-12联合免疫BALB/c小鼠,以ELISA测定小鼠血清中抗HPV16 E5 IgG水平、小鼠脾细胞培养上清中IFN-γ和IL-4含量;MTT法检测脾淋巴细胞增殖反应。结果显示末次免疫后,联合基因疫苗组和单基因疫苗组血清IgG A450值分别明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);且联合基因疫苗组显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组的IFN-γ和IL-4含量分别均明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组IFN-γ和IL-4含量(P<0.01),且联合基因疫苗组含量显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别显著高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P>0.05)。结果表明HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的免疫应答,且联合基因疫苗优于单基因疫苗。     

紫外灭活CVB3 病毒诱导BALB/c小鼠产生免疫保护作用的研究

目的:探讨紫外灭活型CVB3病毒诱导BALB/c小鼠产生特异性免疫应答及保护作用的评估。方法:采用紫外灭活的方法处理野生型CVB3m株,按照0.1 LD50(10~4 PFU)的剂量免疫小鼠,设置PBS免疫组作为对照,免疫后第3,5,7天收集小鼠血清,检测细胞因子含量;在第3,5,7,14天分离小鼠脾脏,流式分析T细胞亚群的分布比例;在免疫后一个月分离小鼠血清,检测中和抗体的滴度;同时间给予小鼠100LD 50野生型CVB3感染,观察小鼠的死亡率。结果:与对照组相比,在检测日期内紫外灭活型CVB3组小鼠血清中细胞因子IL-1α,TNF-α,IL-6的表达量明显增高(P0.05),IL-4的表达量没有明显差异;免疫后第14天CD3~+CD4~+T细胞的分布较对照组明显升高(P0.05);在免疫后一个月,紫外灭活型CVB3免疫组可以诱导机体产生高滴度中和抗体,同时,小鼠应对高致死量CVB3感染时有较高的存活率。结论:紫外灭活型CVB3感染能诱导机体产生特异性免疫应答,同时,产生的中和抗体可以提高小鼠应对致死剂量CVB3感染时的生存率,对机体有明显的保护作用。     

Subcellular components of probiotics Kocuria SM1 and Rhodococcus SM2 induce protective immunity in rainbow trout (Oncorhynchus mykiss, Walbaum) against Vibrio anguillarum

The efficacy of cellular components of probiotics Kocuria SM1 and Rhodococcus SM2 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against vibriosis was assessed. Groups of fish (average weight = 10-15 g) were immunized intraperitoneally (i.p.) with 0.1 ml of subcellular materials, i.e., 0.2 ± 0.05 mg protein per fish, comprising extracellular proteins (ECPs), cell wall proteins (CWPs) and whole cell proteins (WCPs) of SM1 and SM2, respectively, or with 0.1 ml of phosphate-buffered saline (PBS) to serve as the control. Seven days after administration, fish from each group were challenged i.p. with 0.1 ml of a suspension in PBS of 3 × 10(5) cells ml(-1) per fish of Vibrio anguillarum. Use of CWPs and WCPs demonstrated significantly (P < 0.05) better protection against V. anguillarum insofar as mortalities were reduced to 11-17% [relative percent survival (RPS) = 80-87%], although ECPs fared less well (mortalities = 33-38%; RPS = 56-62%; P > 0.05), compared to 86% mortalities of the controls. The mode of action reflected activation of innate immune factors by CWPs and WCPs, demonstrating significantly (P < 0.05) increased expression of respiratory burst (optical density; OD(550 nm)) from 0.039 to 0.043-0.045, peroxidase (OD(550 nm)) from 0.26 to 0.37-0.55, and bacterial killing activities (i.e., percentage of surviving bacteria reduced from 79% to 56-57% for SM2). Moreover, an elevation of leucocyte number (from 1.93% to 1.98-2.93%; P > 0.05) and immunoglubolin level (from 27 mg ml(-1) to 28.5-33 mg ml(-1); P > 0.05) were observed with the experimental groups. These results indicate that cell components of the probiotics stimulate an immune response.     

NLRP3炎症小体对克雷伯杆菌肺炎小鼠肺脏病理损伤的调节作用

摘要 目的：探讨含NLR家族PYRIN域蛋白3（NLR family pyrin domain containing 3,NLRP3）炎症小体对克雷伯杆菌肺炎小鼠肺脏病理损伤的调节作用。方法：56只C57BL/6小鼠随机平分为两组-模型组与对照组,模型组小鼠通过气管注射肺炎克雷伯杆菌建立克雷伯杆菌肺炎模型,对照组小鼠注射等体积的生理盐水,记录与观察肺脏病理损伤情况。结果：模型组建模第7 d与第14 d的肺泡灌洗液髓过氧化酶（Myeloperoxidase,MPO）活性都高于对照组（P<0.05）。模型组建模第7 d与第14 d的肺脏、脾脏、肝脏系数与肺脏病理评分、NLRP3蛋白相对表达水平都高于对照组（P<0.05）。在模型组中,建模第14 d的NLRP3蛋白相对表达水平与肺脏病理评分、肺脏系数、脾脏系数、肝脏系数、肺泡灌洗液MPO活性都存在正相关性（P<0.05）。结论：克雷伯杆菌肺炎小鼠NLRP3炎症小体呈现高表达状况,可介导小鼠肺脏病理损伤,促进MPO活性增加,加重多脏器损伤。