乳酸菌作为传递抗原物质的基因工程疫苗载体的研究进展

乳酸菌是一类重要的食品级工业菌株。将乳酸菌作为传递抗原物质的基因工程载体制备口服疫苗可激发黏膜局部免疫反应和系统免疫反应,增强对胃肠道、呼吸道、生殖道和乳腺病原微生物的抗感染能力,有效预防普通疫苗不能完全解决的传染病。对乳酸菌口服疫苗的研究是近年来的热点,已在此方面取得了突破性成果。     

腺病毒载体狂犬病疫苗研究进展

狂犬病是一种古老的人兽共患传染病。世界上已有部分国家和地区成功地消灭了狂犬病。在我国,每年有约3 000人死于狂犬病。腺病毒作为载体的优点包括高效的入核机制、优良的转染性、较低的病原性、较高的基因表达量及清楚的基因背景。目前,腺病毒已广泛应用于基础研究、基因治疗和疫苗研发。20世纪末,以腺病毒为载体的表达狂犬病病毒糖蛋白的疫苗就已诞生,一直发展至今。以腺病毒载体狂犬病疫苗作为口服疫苗,可望在控制和消除狂犬病中发挥着巨大的作用。     

口服疫苗载体--可生物降解微粒的研究进展

口服疫苗因其安全、应用方便、可避免因注射引发的交叉感染而倍受青睐,现已成为疫苗研究的热点之一,适合的抗原载体系统是制约其研制和开发的关键,而可生物降解微粒为解决这一问题开辟了新领域。1 制备可生物降解微粒的材料及其优点     

口服酵母载体疫苗在畜禽疫病防控中的应用

酵母载体疫苗由于其安全性、免疫方式、生产成本等方面的显著优势,近年来受到关注。酵母细胞壁含有的β-葡聚糖可作为天然佐剂;通过将目的蛋白锚定在酵母细胞表面制成酵母表面展示疫苗、表达在胞内的全重组酵母疫苗口服免疫使用,不仅可激发机体黏膜免疫,调节微生物菌群,还可省去蛋白质纯化、免疫注射等流程,在降低人工成本,促进无抗化健康养殖方面具有重要助推作用。论文简述了酵母疫苗表达系统及其优势、免疫机制及生产应用现状,展望了酵母表达系统在动物疫病防控中的潜在应用。     

药物递送载体及其在饲料添加剂领域中的研究进展

在全面禁止使用促生长类药物饲料添加剂的形势下,畜禽传染性疾病对我国畜牧生产造成潜在威胁,开发绿色饲料添加剂迫在眉睫,但当下抗生素替代产品往往因稳定性差而限制了其使用空间.药物递送系统可以将兽药及饲料添加剂包裹于载体内部,靶向缓释,具有靶向性、稳定性及高效性等多方面的优势,是解决粪便抗生素残留、添加剂稳定性差等问题的有效...     

纳米材料作为抗菌肽递送载体的研究进展

由于抗生素的过度使用和滥用,抗生素耐药性也随之产生,抗菌肽作为新型抗菌剂可减少抗生素的使用.随着对抗菌肽研究的深入,发现低蛋白酶抗性、溶血性以及不稳定性等缺点使其在临床应用上受限.除了在结构上对抗菌肽进行修饰和改造外,使用纳米材料作为递送系统传递抗菌肽也可以改善其不足并提高治疗效果.纳米材料能够与抗菌肽协同抗菌,对药物...     

动物疫病活载体疫苗研究进展

活载体疫苗是以细菌或病毒作为载体表达外源抗原和治疗因子的载体系统,具有安全性高、毒力返祖风险低、成本低,可诱导免疫机体产生高水平的体液免疫、细胞免疫或黏膜免疫等优点,是目前最具发展潜力的基因工程疫苗之一,在动物疫病防控领域应用较多。病毒载体包括DNA病毒（如腺病毒、腺相关病毒和痘病毒等）和RNA病毒（如新城疫病毒、流感病毒等）;细菌载体包括减毒致病菌与非致病菌两类,主要包括乳酸菌、沙门氏菌、大肠杆菌等。活载体疫苗常用的抗原呈递策略有载体-宿主平衡致死系统、微生物表面展示系统。多种疫苗载体的开发及抗原呈递策略的选择,使得活载体疫苗的使用价值最大化。不同载体疫苗在预防疫病方面均有不同优缺点,应根据实际情况选择最优最适合的活载体疫苗。本文综述了动物疫病防控领域的病毒和细菌活载体疫苗研究进展及其抗原呈递方式,以期为活载体疫苗的进一步研究提供参考。     

转基因植物口服疫苗研究进展

利用转基因技术构建植物生物反应器,生产口服疫苗是目前新兴的研究领域。与其他疫苗生产方式相比,转基因植物口服疫苗生产技术具有高效、经济和简便等特点。根据转基因植物口服疫苗的特点,人和家畜家禽摄食方式的不同,应用转基因植物口服疫苗预防集约化饲养动物传染病的发生和流行,具有更为重要的意义。因此,转基因植物口服疫苗的研究热点逐渐由人的转基因植物口服疫苗向家畜和家禽的转基因植物口服疫苗的方向转移。文章就转基因植物口服疫苗的优点、人口服的转基因植物疫苗、家畜饲用的转基因植物疫苗及禽用的转基因植物口服疫苗做一综述。     

重组羊奇异变形杆菌ompA乳酸乳球菌的构建及其在小鼠体内的定植规律检测

构建了含羊奇异变形杆菌外膜蛋白A(ompA)编码基因的重组载体pNZ8149-ompA,并将其电转入乳酸乳球菌NZ3900中。利用Westen blot检测目的蛋白的表达,流式细胞仪及间接免疫荧光技术检测目的蛋白在乳酸乳球菌中的定位。将重组乳酸乳球菌灌胃BALB/c小鼠,分别于口服后1,2,3,4,5,6,7d取其十二指肠、空肠、回肠、盲肠的肠道冲洗液,通过平板菌落计数法检测乳酸乳球菌在肠道的定植情况。Western blot检测到目的蛋白于49 000处出现特异性条带;流式细胞仪检测到重组乳酸乳球菌表面荧光强度明显强于空质粒对照组;间接免疫荧光试验检测到重组乳酸乳球菌菌体表面出现绿色荧光。灌服小鼠后,重组乳酸乳球菌在小鼠各肠段的定植比例不同,定植能力于口服后6d达到高峰,7d后在重组菌在十二指肠、空肠、回肠和盲肠的定植率分别占第1d的15.23%,24.19%,48.57%,40.07%。本试验证明羊奇异变形杆菌ompA能够稳定表达于乳酸乳球菌表面,且重组乳酸乳球菌在小鼠肠道内具有良好的定植能力,其定植能力为回肠盲肠空肠十二指肠。这为研究乳酸乳球菌作为羊奇异变形杆菌口服疫苗抗原递送载体提供了试验基础。     

动物寄生虫口服疫苗研究进展

寄生虫病严重影响全球人类、动物的健康安全,所带来的经济损失巨大。因此,有必要研制针对寄生虫病的疫苗,以阻断寄生虫病在动物与动物、动物与人类之间的传播。寄生虫存在多种免疫逃避机制,已开发的亚单位疫苗、减毒活疫苗、灭活疫苗均未达到理想的预防效果,且商品化疫苗多为针剂疫苗,普通针剂疫苗操作繁琐、运输储藏要求高且易使动物发生应激,直接影响经济成本,因此在实际生产当中需更多操作简单、便于储存、免疫成本更低的新型疫苗,以有效防控寄生虫病。诸多研究表明口服疫苗操作方便,只需通过口服方式投喂且宿主获得的抗体效价水平较高,有望成为预防寄生虫病的有效手段。口服疫苗是一种新型疫苗,依靠宿主的黏膜免疫系统来产生作用,即通过机体黏膜表面接种便可同时诱导机体产生持久的黏膜免疫、体液免疫和细胞免疫,为宿主提供高效的免疫保护。与传统疫苗相比,口服疫苗最显著的优点就是便于接种且应激小,还能形成强大的黏膜免疫屏障。但胃肠道的环境及易形成免疫耐受等因素也为口服疫苗的开发带来很大的挑战。笔者就黏膜免疫系统与口服疫苗作用机理、近几年寄生虫口服疫苗的研究进展及优点与挑战进行综述,以期为寄生虫口服疫苗的开发提供理论依据。     

产肠毒素大肠杆菌K88黏附素蛋白亚基Fae G在L.lactis中的表达及其抗原性

在构建L.lactis表达载体pNZ8148-fae G的基础上,将该载体转化到L.lactis NZ9000中,重组菌经5 μg/Lnisin诱导3 h,SDS-PAGE结果显示,FaeG在L.lactis中表达的目的蛋白相对分子质量大小约为27 000,表达产物的量达菌体可溶性总蛋白的10.89%.Western blot分析结果表明,该目的蛋白具有良好反应原性.将重组L.lactis口服免疫BALB/c小鼠,小鼠产生特异性IgG及黏膜slgA.本试验为进一步研究仔猪口服重组L.lactis疫苗,诱导其产生抗ETEC K88黏膜免疫的同时,发挥L.lactis的益生功能,预防仔猪腹泻奠定了基础.     

转基因植物生产疫苗的研究现状及前景

对转基因植物作为生物反应器生产可食疫苗的作用机理、优点、可行性及研究现状进行概述,指出了转基因植物疫苗研究面临的问题与前景。     

Polymeric nanoparticles as an oral delivery system for biocontrol agents for the brushtail possum (Trichosurus vulpecula)

Abstract

AIM: To investigate polymeric nanoparticles as an oral delivery system for protein biocontrol agents for control of the brushtail possum, Trichosurus vulpecula.

METHODS: Insulin-loaded poly(ethyl 2-cyanoacrylate) (PECA) nanoparticles were prepared using interfacial polymerisation, and characterised for size, zeta potential, and efficiency of encapsulation of insulin. In-vitro release of insulin-loaded PECA nanoparticles was quantified using reverse-phase highpressure liquid chromatography (HPLC). The in-vivo pharmacokinetics of insulin in PECA nanoparticles was investigated following I/V administration, and when injected directly into the caecum alone or in conjunction with the permeation enhancer EDTA. Blood samples were collected at intervals from ?5 to 180 minutes, and the concentration of insulin in plasma was quantified using a radioimmunoassay (RIA) validated for possum plasma.

RESULTS: Poly(ethyl 2-cyanoacrylate) nanoparticles were produced with a uniform particle size of 200–300 nm, and the mean entrapment of insulin was 78%. In-vitro release of insulin from the PECA nanoparticles was controlled, although incomplete, and approximately 30% of the insulin remained entrapped. The bioavailability of insulin when administered in a PECA nanoparticulate formulation injected directly into the caecum was <1%, and was not increased by addition of the permeation enhancer.

CONCLUSIONS: The nanoparticulate formulations investigated as part of this study resulted in low bioavailability of the peptide insulin in the brushtail possum.     

The effect of diethylene glycol monoethyl ether as a vehicle for topical delivery of ivermectin

The effect of diethylene glycol monoethyl ether (DGME; Transcutol) on the permeation of ivermectin, a broad-spectrum antiparasitic agent, through bovine skin was evaluated byin vitro permeation experiments followed by serial sectioning of the skin to assess the amount of ivermectin retained in the skin. Ivermectin permeation through bovine skin was enhanced by DGME and this enhancement was DGME-concentration-dependent. Permeation of ivermectin was effectively enhanced in vehicles with low proportions of DGME, but the magnitude of permeation enhancement decreased as the proportion of DGME increased. The permeation was accompanied by the formation of cutaneous depots of ivermectin. Furthermore, the data indicated that the flux and the cutaneous accumulation of ivermectin were sensitive to the concentration gradient of DGME across the skin. This suggested that ivermectin was permeating with DGME, in which it is very soluble. Hence, the enhancing mechanism involves solubilization of the ivermectin by DGME and the transport of DGME itself across the skin. Based on these results, DGME appears to be a potential vehicle for topical delivery of ivermectin by transport through the skin and through formation of cutaneous depots of ivermectin.Abbreviations DGME diethylene glycol monoethyl ether     

Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Δlon) and JOL916 (ΔlonΔcpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (ΔcpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD₅₀ using 1-week-old chickens, were attenuated approximately at 10¹-, 10¹-, and > 10³-fold, respectively. The oral inoculations of 2 × 10⁷ cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT.     

Quantitative real‐time PCR monitoring of Escherichia coli and Clostridium perfringens with oral administration of Lactobacillus plantarum strain Lq80 to weaning piglets

Levels of fecal or intestinal lactobacilli, Escherichia coli and Clostridium perfringens, and the prevalence of clostridial alpha toxin gene and heat‐stable toxin (ST) gene of enterotoxigenic E. coli (ETEC) were monitored in weaned piglets before (day 0) and during (days 7, 14, and 21) the administration of Lactobacillus plantarum strain Lq80. Lactobacilli were enumerated in a culture‐dependent method. The remainders were determined by quantitative real‐time PCR. In this quantitative real‐time PCR method, the detection limit was proved to be as low as 10³ cells/g feces or intestinal contents. Number of lactobacilli increased from day 0 to day 7 (P < 0.05), to day 14 (P < 0.05), and to day 21 (P = 0.07) in the Lq80‐administered group. L. plantarum contributed to as low as 10% of the lactobacillal population in the Lq80‐administered group. The number of E. coli and C. perfringens, and the prevalence of alpha toxin gene in feces or intestinal contents of the Lq80‐administered group decreased, at least in the first week of the postweaning period. Oral administration of L. plantarum strain Lq80 can stimulate the growth of indigenous lactobacilli and decrease ST‐producing ETEC and C. perfringens in the intestine of postweaning piglets.     

Potent immune responses induced by a Salmonella ghost delivery system that expresses the recombinant Stx2eB,FedF, and FedA proteins of the Escherichia coli-producing F18 and Shiga toxin in a murine model and evaluation of its protective effect as a porcine vaccine candidate

Background: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18⁺ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen.

Objective: To develop a vaccine candidate against ED by combining three selected antigens of F18⁺ E. coli.

Methods: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay.

Results: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3⁺CD4⁺ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls.

Conclusion: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.