重组CRM197-4GnRH去势疫苗抗原分子的原核表达及免疫效果评价

通过大肠杆菌表达系统对CRM₁₉₇-4GnRH重组去势疫苗抗原进行表达,采用不同种类佐剂制备疫苗,研究其对大鼠的免疫去势作用,为获得1种免疫效果好、有效期长、生产成本低的促性腺激素释放激素(GnRH)免疫去势基因工程疫苗提供借鉴。采用白喉毒素无毒突变体(CRM₁₉₇)与4拷贝GnRH串联,将CRM₁₉₇-4GnRH基因与表达载体连接,并通过优化试验确定大肠杆菌的最优表达条件,用SDS-PAGE和Western blot鉴定目标蛋白的表达情况。将融合蛋白与不同佐剂乳化,制备成9种测试疫苗后分别免疫大鼠,测定血清中GnRH抗体滴度和睾酮浓度。结果显示,重组大肠杆菌的最优表达条件为诱导时间表达5 h,培养基为LB液体培养基,IPTG诱导浓度为0.5 mmol/L;通过SDS-PAGE和Western blot试验,表明目标融合蛋白已成功表达;动物试验结果表明,重组疫苗水包油包水佐剂组、重组疫苗油包水佐剂组、GnRH+Th抗原(辅助型T细胞抗原)表位(G1抗原)油包水佐剂组测试疫苗免疫大鼠较其他组GnRH抗体滴度显著升高、睾酮浓...     

不同佐剂GnRH疫苗主动免疫对公猪睾丸发育和血清睾酮的影响

为探讨铝胶佐剂与白油Span佐剂的GnRH疫苗对公猪睾丸发育和血清睾酮的影响,12头初生公猪随机分为GnRH疫苗铝胶佐剂免疫组和GnRH疫苗白油Span佐剂免疫组(下简称铝胶组和白油组).9周龄初免,17周龄加强免疫,25周龄屠宰.游标卡尺测量猪阴囊直径和睾丸大小,石蜡切片观察睾丸组织发育,ELISA法和放射免疫分析(RIA)法分别检测GnRH抗体水平和血清睾酮水平.结果表明,铝胶组与白油组GnRH抗体效价差异不显著(P0.05).铝胶组血清睾酮水平于17,21周龄(P＜0.05)和25周龄(P＜0.01)显著地低于白油组,阴囊直径,睾丸直径、横径、周长及睾丸血量均显著地低于白油组(P＜0.05).铝胶组与白油组猪睾丸曲精小管均出现不同程度的空泡化,白油组零星分布少量精子,铝胶组无精子产生.这表明在公猪免疫去势的效果上,辅以铝胶佐剂的GnRH疫苗比辅以白油Span佐剂的GnRH疫苗好.     

蜂胶———一种优质的动物疫苗佐剂

疫苗佐剂是一种免疫增强剂,自身并不能引起机体产生免疫应答,但能提高抗原的免疫原性和免疫反应的可持续性,诱发机体针对特定抗原产生更加长期、高效的免疫应答反应,增强疫苗对机体的免疫保护作用。随着人们对动物食品卫生安全的关注,绿色、环保、健康的理念已逐渐深入人心,普遍将“使用要安全、免疫要高效、抗体产生快、免疫保护强”这几点作为动物疫苗佐剂研究的方向。蜂胶是由蜜蜂的工蜂采集杨树、柳树、松树等植物的幼芽等同自身的消化腺和蜡腺等分泌物进行混合之后形成的一种具有芳香气味的胶状固体物,含有黄酮类、萜烯类、有机酸、芳香醛、醇类、酯类及多种氨基酸、酶、维生素、矿物质等,其主要的成分为黄酮类化合物,具有抗菌、抗病毒、抗肿瘤、消炎、增强机体免疫功能和促进组织再生等广泛的生物学作用,是良好的动物疫苗佐剂。早在20世纪90年代,我国沈志强等研究人员以蜂胶为免疫佐剂,开创了疫苗佐剂研究的新领域,使我国蜂胶佐剂疫苗的研究与应用处于世界领先地位。20年来,蜂胶在动物疫苗佐剂中得到迅速发展和广泛应用,论文着重介绍了蜂胶在猪疫苗佐剂、禽疫苗佐剂、兔疫苗佐剂、犬疫苗佐剂中的应用研究进展及应用概况,旨在为蜂胶在动物疫苗佐剂中的开发利用与推广应用积累资料。     

家畜免疫去势研究进展

传统的手术去势技术正受到动物福利和规模化标准化畜牧生产需求的挑战,作为手术去势的优势替代方法,免疫去势能够很好地阻滞睾丸发育,控制性行为,避免手术带来的痛苦。免疫去势是采用免疫学方法破坏下丘脑-垂体-性腺轴的激素平衡,通过降低促性腺激素释放激素（GnRH）、黄体生成素（LH）和卵泡刺激素（FSH）的水平,进而使性腺激素水平降低,最终抑制性腺功能,达到去势的目的。免疫去势分为激素免疫去势和基因免疫去势,其靶标经历了下丘脑-垂体-性腺轴自下而上的筛选,依次为睾酮、LH、FSH、GnRH、吻素-1（KISS-1）,其中位于下丘脑-垂体-性腺轴上游的GnRH激素、GnRH和KISS-1基因3种靶标去势效果较好。免疫去势效果及机制的相关研究表明,免疫去势具有改善生产性能、安全和可逆的特点,但其可逆性机制还不清楚。去势导致肾上腺发挥代偿作用,但目前免疫去势的肾上腺代偿研究鲜见报道。文章首先介绍了几种靶标的免疫去势机制,其次对免疫去势的特点和应用进行了讨论,旨在为推动免疫去势技术在动物生产繁殖领域的应用提供理论依据。     

DNA疫苗佐剂研究进展

DNA疫苗是近年来发展较为迅速的一类生物制剂,它能诱导动物机体产生持久的体液免疫和细胞免疫,其在抗病毒、细菌和寄生虫的感染方面起到了重要作用。但与传统的灭活疫苗相比,其免疫效果并不理想。为了攻克DNA疫苗免疫效力低下的难题,一些学者已经研制了多种佐剂制品,以提高DNA疫苗对动物疾病免疫保护的能力。文章对使用佐剂提高DNA疫苗免疫效力进行了综述。     

在液体代乳日粮和干日粮中添加左旋精氨酸可提高21日龄断乳后头3周的仔猪生产性能

本研究的目的是评价GnRH（促性腺激素释放激素）疫苗——Improvac（TM）（Pfizer）抑制公猪攻击行为和性行为的效果。136头猪分为3组：完整公猪组（64头，即非去势非免疫组），抗GnRH免疫去势组（48头）和手术去势组（24头）。     

家畜免疫去势研究进展

传统的手术去势技术正受到动物福利和规模化标准化畜牧生产需求的挑战,作为手术去势的优势替代方法,免疫去势能够很好地阻滞睾丸发育,控制性行为,避免手术带来的痛苦。免疫去势是采用免疫学方法破坏下丘脑-垂体-性腺轴的激素平衡,通过降低促性腺激素释放激素(GnRH)、黄体生成素(LH)和卵泡刺激素(FSH)的水平,进而使性腺激素水平降低,最终抑制性腺功能,达到去势的目的。免疫去势分为激素免疫去势和基因免疫去势,其靶标经历了下丘脑-垂体-性腺轴自下而上的筛选,依次为睾酮、LH、FSH、GnRH、吻素-1(KISS-1),其中位于下丘脑-垂体-性腺轴上游的GnRH激素、GnRH和KISS-1基因3种靶标去势效果较好。免疫去势效果及机制的相关研究表明,免疫去势具有改善生产性能、安全和可逆的特点,但其可逆性机制还不清楚。去势导致肾上腺发挥代偿作用,但目前免疫去势的肾上腺代偿研究鲜见报道。文章首先介绍了几种靶标的免疫去势机制,其次对免疫去势的特点和应用进行了讨论,旨在为推动免疫去势技术在动物生产繁殖领域的应用提供理论依据。     

GnRH免疫对家畜生殖调控的研究进展

GnRH免疫去势技术是近十多年来兴起的一项简单易行、效果良好的动物繁殖调控技术,现已在多种动物去势及避孕方面取得了较大发展。论文较为全面的介绍了GnRH的结构及生物学功能、GnRH免疫的作用机制及其在动物生殖调控中的应用,并对GnRH免疫的前景进行了展望,以期为进一步研究GnRH免疫提供了一定的理论指导。     

GnRH二聚体和GnRH并列二聚体合成肽疫苗对大鼠的免疫去势效果

为了比较GnRH二聚体和GnRH并列二聚体单次和两次免疫对雄性大鼠的免疫去势效果。将上述两种GnRH疫苗,分组免疫SD大鼠,免疫后每两周检测大鼠血清中的GnRH抗体滴度和血清睾酮水平、睾丸重量和组织结构变化。结果显示,两次免疫试验组的抗体滴度显著高于单次免疫试验组的抗体滴度(P<0.05);GnRH二聚体抗原免疫效果优于GnRH并列二聚体抗原的免疫效果,且差异极显著(P<0.01)。免疫组大鼠血清睾酮水平及睾丸重量均极显著低于空白对照组(P<0.01)。免疫去势效果显著的大鼠只数统计显示,GnRH二聚体(5/6)和GnRH并列二聚体(4/6)两次免疫均多于单次免疫(4/6和3/6)。同时,组织学观察结果可见,免疫组大鼠睾丸组织结构萎缩,曲精小管管径变窄,内部精母细胞脱落,精子变少或者没有。表明GnRH二聚体疫苗与GnRH并列二聚体疫苗均能达到去势效果,前者优于后者,两次免疫优于一次免疫。     

使用不同佐剂去势疫苗免疫效果的比较

分别应用明矾、氢氧化铝和弗氏佐剂的去势疫苗免疫 SD大鼠 ,检测大鼠 LHRH抗体效价、睾酮水平、观察睾丸肉眼及组织学变化 ,以及精子数量、畸形率的变化 ,确定去势疫苗的效果。结果表明明矾、氢氧化铝和弗氏佐剂组均能够较好的诱导大鼠产生 L HRH抗体 ,引起睾酮明显降低。三个免疫组与阴性对照组相比 ,都能使大鼠明显增重 ,并起到萎缩性腺的作用     

GnRH并列体二聚体主动免疫对雄兔血清睾酮的影响

用GnRH并列体二聚体主动免疫雄兔,并观察了免疫对雄兔血清睾酮与睾丸重量的影响。结果表明该方法能使雄兔血清睾酮水平降低,睾丸重量下降。     

The function of the pituitary-testicular axis in dogs prior to and following surgical or chemical castration with the GnRH-agonist deslorelin

Chemical castration, that is the reduction of circulating testosterone concentrations to castrate levels by administration of a GnRH-agonist implant, is a popular alternative to surgical castration in male dogs. Detailed information concerning the pituitary-testicular axis following administration of a GnRH-agonist implant is still scarce. Therefore, GnRH-stimulation tests were performed in male dogs, prior to and after surgical and chemical castration. This approach also allowed us to determine plasma concentrations of testosterone and oestradiol in intact male dogs for future reference and to directly compare the effects of surgical and chemical castration on the pituitary-testicular axis. In intact male dogs (n = 42) of different breeds GnRH administration induced increased plasma LH, FSH, oestradiol and testosterone concentrations. After surgical castration basal and GnRH-induced plasma FSH and LH concentrations increased pronouncedly. Additionally, basal and GnRH-induced plasma oestradiol and testosterone concentrations decreased after surgical castration. After chemical castration, with a slow-release implant containing the GnRH-agonist deslorelin, plasma LH and FSH concentrations were lower than prior to castration and lower compared with the same interval after surgical castration. Consequently, plasma oestradiol and testosterone concentrations were lowered to values similar to those after surgical castration. GnRH administration to the chemically castrated male dogs induced a significant increase in the plasma concentrations of LH, but not of FSH. In conclusion, after administration of the deslorelin implant, the plasma concentrations of oestradiol and testosterone did not differ significantly from the surgically castrated animals. After GnRH-stimulation, none of the dogs went to pre-treatment testosterone levels. However, at the moment of assessment at 4,4 months (mean 133 days ± SEM 4 days), the pituitary gonadotrophs were responsive to GnRH in implanted dogs. The increase of LH, but not of FSH, following GnRH administration indicates a differential regulation of the release of these gonadotrophins, which needs to be considered when GnRH-stimulation tests are performed in implanted dogs.     

兽用疫苗中药佐剂发展与管理研究

本文聚焦兽用疫苗中药佐剂的研究与产业化发展，总结兽医领域在单味药与中药组份佐剂，以及配合疫苗使用的“免疫增强剂”效用，从现代免疫与中兽医理论两个角度分析作用机理，推动兽用疫苗中药佐剂在提高免疫效果、提高疫苗免疫应答方面的应用，提出产业发展对策与管理建议。     

Effect of active immunization against GnRH on androstenone concentration, growth performance and carcass quality in intact male pigs

The objective of this study was to investigate the efficacy of active immunization against gonadotropin releasing hormone (GnRH) in male pigs and to compare it with surgical castration. Piglets were randomly assigned to two groups, one of 263 animals for surgical castration (SC) and one of 270 animals for immunocastration (IC). Surgery was done at 14 days of age and vaccination (Improvac®, CSL, Australia) performed twice at an interval of 4-5 weeks with the second injection given 4-7 weeks before slaughter. At slaughter, testes were weighed and fat samples collected for androstenone analysis. Androstenone was tested olfactorially by heating salivary glands in a microwave oven. Daily growth rate and meat quality were assessed in all animals. Regarding mean androstenone concentrations in backfat, no significant difference was found between SC and IC (0.042 (0.041; 0.044) μg/g vs. 0.058 (0.044; 0.071) μg/g; mean (95% confidence interval)). Testes weight was significantly (P<0.001) smaller in immunized (230.8 g (218.23; 243.52)) as compared to normal boars (761.8 g (722.77; 801.01)) of the same age, as a reference. In this study, androstenone production was suppressed in all vaccinated animals and the carcasses given free for consumption. Mean daily growth gain was not significantly different between SC (0.817 kg (0.804; 0.830)) and IC (0.827 kg (0.814; 0.840)), although there was a trend for better performance in the latter group. The yield of lean meat was significantly (P<0.001) improved in IC (54.50% (54.26; 54.73)) when compared to SC (53.76% (53.53; 54.00)). From our results, we conclude that vaccination against GnRH is a practical and effective method to suppress androstenone synthesis. Pain and stress associated with surgical castration can thus be avoided.     

GnRH-Ⅰ疫苗免疫效果影响因素、商业困境与免疫机理研究进展

哺乳动物中除豚鼠外GnRH-Ⅰ的主要序列和结构都是完全一致的，所以GnRH-Ⅰ疫苗的应用范围广。在动物去势及避孕和人类免疫治疗研究中显示了其无可比拟的优越性。本文综述了GnRH-Ⅰ疫苗的免疫机理、免疫效果的影响因素及其商业化的困境。     

Aggressive behavior is reduced in bulls actively immunized against gonadotropin-releasing hormone

The purpose of this research was to compare the frequency of aggressive behavior's in beef bulls actively immunized against gonadotropin-releasing hormone relative to contemporary nonimmunized control bulls and surgically castrated steers. Eight males were assigned to each ofthese treatments in each of 4 yr. Immunized males were treated with a GnRH-keyhole-limpet hemocyanin (KLH) conjugate at approximately 4 mo of age. A secondary (booster) immunization was administered at 12 mo. Steers were castrated at 4 mo of age. Animals in each treatment in each year were housed as a single group prior to testing. At approximately 16 mo of age, each group of eight animals was placed in a 10- x 16-m enclosure for 20 min on five occasions at 2 to 3 d intervals. An observer recorded butts initiated by each animal as well as participation in bouts of sparring. Relative to control bulls, immunocastration reduced the frequency of butts initiated (P < 0.05) and participation in sparring bouts (P < 0.05) to levels typically observed in steers (P > 0.05). These observations indicate that active immunization against GnRH reduces the incidence of aggressive behavior in male beef cattle and are consistent with our postulate that immunoneutralization of GnRH is an effective alternative to surgical castration in the management of beef cattle.     

Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model

Enteric viruses are a major cause of diarrhea in animals and humans. Among them, rotaviruses are one of the most important causes of diarrhea in young animals and human infants. A lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses. Studies of candidate vaccines using an adult mouse model of subclinical enteric viral infections often do not predict vaccine efficacy against disease evaluated in neonatal large animals. A series of studies have been conducted using a neonatal gnotobiotic pig model of rotavirus infection and diarrhea to identify correlates of protective immunity and to evaluate traditional and novel vaccine approaches for the induction of mucosal immune responses and protection to enteric viruses. Gnotobiotic pigs recovered from infection with virulent Wa human rotavirus (HRV) (mimic natural infection) had high numbers of intestinal IgA rotavirus-specific primary antibody-secreting cells (ASCs) and memory B-cells (to recall antigen) measured by ELISPOT assay, which correlated with complete protection against rotavirus challenge. Most short-term IgA memory B-cells were resident in the ileum, the major site of rotavirus replication. Spleen, not the bone marrow, was the major resident site for longer-term IgG memory B-cells. Candidate rotavirus vaccines evaluated in pigs for their ability to induce intestinal or systemic ASC and protection against rotavirus infection and diarrhea included attenuated live virus, inactivated virus, and baculovirus-expressed double-layered rotavirus-like particles (2/6-VLPs). In combination with those candidate vaccines, various adjuvants, delivery systems, and immunization routes were tested, including incomplete Freund's adjuvant for i.m. immunization, and a mutant Escherichia coli heat labile enterotoxin R192G (mLT) for i.n. immunization. It was shown that orally administered replicating vaccines were most effective for priming for intestinal IgA ASC and memory B-cell responses, but i.n. administered non-replicating 2/6-VLPs plus mLT were effective as booster vaccines. We conclude that protective immunity depends on the magnitude, location, viral protein-specificity, and isotype of the antibody responses induced by vaccination. Therefore highly effective enteric viral vaccines should: (i) induce sufficient levels of intestinal IgA antibodies; (ii) include viral antigens that induce neutralizing antibodies; and (iii) require the use of effective mucosal adjuvants or antigen delivery systems for non-replicating oral or i.n. vaccines.     

淫羊藿多糖对雏鸡免疫器官及新城疫疫苗免疫效果的影响

疫苗免疫接种是预防和控制传染病的重要措施,而佐剂是疫苗研究的重要组成部分.选择合适的佐剂不仅可以增强疫苗的免疫效果,而且可以提高机体的抵抗力.本研究将50、100、150 g/L等不同浓度的淫羊藿多糖与灭活的鸡新城疫病毒乳化,制备疫苗免疫雏鸡,通过检测抗体效价和免疫器官指数,探讨淫羊藿多糖作为免疫佐剂对鸡免疫功能的影响.结果表明,100 g/L和150 g/L的淫羊藿多糖作为免疫佐剂均可促进抗体的产生,提高脾脏指数,但对法氏囊指数的促进作用不明显.     

Active Immunization against Gonadotrophin-releasing Hormone in Chinese Male Pigs

We have investigated, under the normal conditions of local Chinese pig farming, castration of young male pigs by vaccination with a newly developed vaccine against gonadotrophin releasing hormone (GnRH). Because of the very early onset of puberty, long fattening period and relatively harsh circumstances in Chinese pig production, an investigation of the endocrine response of Chinese breeds to this type of vaccination was of particular interest. Fifteen crossbred boars (Yorkshire × Yanan) from three different litters were randomly assigned to three groups of five animals each. The first group was immunized at 13 weeks of age with a GnRH tandem dimer OVA‐conjugate in Specol and received a booster immunization 8 weeks later. The second group was injected with Specol alone and served as untreated controls. The remaining group was surgically castrated at the time of weaning (at 6 weeks of age). Pigs were fed ad libitum from weaning onwards. All animals were slaughtered at 31 weeks of age. Immunized boars had undetectable or low serum testosterone (0.09 ± 0.12 ng/ml), low fat androstenone (0.05 ± 0.01 μg/g) levels and very low testes weights (19.1 ± 4.3 g). Intact controls had much higher serum levels of testosterone (9.76 ± 4.81 ng/ml), fat androstenone levels (2.26 ± 0.87 μg/g) and testes weights (114.3 ± 29.41 g) at slaughter. Both the immunized and castrated group grew significantly faster than intact boars (p < 0.01). Average daily gains in immunized, castrated and intact animals were 0.69 ± 0.08, 0.63 ± 0.05 and 0.42 ± 0.07 kg (mean ± SD), respectively. The present data demonstrate for the first time that the newly developed anti‐GnRH vaccine works very well under practical Chinese pig farming conditions, and can be an attractive alternative to surgical castration.