表达鸡IL-4转基因和缓艾美耳球虫的构建及其繁殖力、免疫原性和对鸡血液白细胞数量的影响

发布时间：2019-01-10 10:21

【摘要】：鸡球虫病是严重危害养鸡业的一种寄生原虫病,由顶复门艾美耳属球虫感染引起,每年给世界养鸡业造成愈20亿美元的经济损失。强毒与弱毒球虫病活疫苗被认为是预防禽球虫病的最有效方法之一。尽管可通过球虫病活疫苗控制鸡球虫病的发生,但由于有些球虫种的免疫原性较差,只能使宿主产生部分免疫保护,致使这些球虫种多次反复感染而负面影响了免疫鸡群的饲料报酬,使得球虫病疫苗在肉鸡上的使用受到了限制。因此,为了更有效的预防鸡球虫病,通过佐剂增强免疫原性较差的虫种的免疫原性变得十分重要。细胞因子如白细胞介素-4(IL-4),对抗原的免疫原性有显著影响,在免疫应答过程中扮演着关键角色。IL-4是Ⅱ型辅助T细胞(Th2细胞)分泌的细胞因子,其生物学作用主要为刺激活化的B细胞和T细胞增殖,诱导未成熟的辅助T细胞分化成Ⅱ型辅助T细胞。IL-4是否有提高球虫免疫原性的功能尚不清楚。本研究构建了一株表达鸡白细胞介素-4(ChIL-4)的转基因和缓艾美耳球虫(EmiChIL-4),研究ChIL-4的佐剂效应及其在抗球虫感染中的作用。首先,我们利用双表达框载体策略构建了表达白细胞介素-4以及表达黄荧光蛋白(YFP)-乙胺嘧啶抗性基因的球虫转染载体,将其以核转染法转染和缓艾美耳球虫子孢子。转染的子孢子一部分接种至鸡肾原代细胞,在荧光显微镜下观察体外瞬时转染效率；另一部分经泄殖腔接种鸡进行药物压力下稳定转染筛选,收集感染后5-10天排出的卵囊,孢子化后进行传代,成功传代7次,获得了稳定的转基因和缓艾美耳球虫虫株。为了比较表达鸡IL-4转基因和缓艾美耳球虫(EmChlL-4)和野生和缓艾美耳球虫(wild type)激发机体产生的免疫应答水平和免疫保护力,我们将12只12日龄的AA肉鸡分成三组,即PBS组、野生和缓艾美耳球虫组、转基因和缓艾美耳球虫组,分别口服接种PBS悬液、孢子化野生和缓艾美耳球虫卵囊、孢子化转基因和缓艾美耳球虫卵囊,两次免疫后进行攻毒。通过测定卵囊排出量、特异性IgY (IgG)抗体滴度等实验方法来比较两种虫株的免疫原性。卵囊排出量测定结果显示：相比野生和缓艾美耳球虫组,转基因和缓艾美耳球虫组的卵囊排出量减少了6倍,排卵囊高峰期延迟了22小时,表明转基因虫株免疫的鸡比野生株免疫的鸡建立了更好的免疫保护。ELISA结果显示转基因组血清中特异性IgY抗体滴度明显高于野生组,表明了相比野生和缓艾美耳球虫,EmChIL-4激发机体产生了更高的体液免疫应答。另外,我们还比较了EmChIL-4与野生母株的卵囊产量及对感染鸡血液常规指数的影响。结果显示感染EmChIL-4 I'的鸡的卵囊产量显著低于感染野生母株的鸡的卵囊产量；且白细胞总数,包括嗜酸性粒细胞、嗜碱性粒细胞和淋巴细胞与感染野生母株的鸡相比明显降低；提示ChIL-4在转基因和缓艾美耳球虫中的表达致弱了和缓艾美耳球虫。综上所述,表达鸡IL-4转基因和缓艾美耳球虫获得了更高的免疫原性和更低的毒力,提示表达细胞因子IL-4的转基因和缓艾美耳球虫有作为高效抗球虫病疫苗的组成成分的潜力,帮助鸡群建立对球虫感染更好的免疫保护。
[Abstract]:The chicken coccidiosis is a parasitic protozoan disease which is seriously harmful to the chicken industry, which is caused by the infection of Eimeria, and the economic loss of the more than $2 billion is caused to the chicken industry every year. The live vaccine of virulent and attenuated coccidiosis is considered to be one of the most effective methods for the prevention of coccidiosis in poultry. Although the occurrence of chicken coccidiosis can be controlled by the coccidiosis live vaccine, because of the poor immunogenicity of some coccidiosis species, the host can only generate partial immune protection, so that the coccidiosis can repeatedly infect and adversely affect the feed compensation of the immune chicken group, so that the use of the coccidiosis vaccine on the broiler is limited. Therefore, in order to be more effective in the prevention of chicken coccidiosis, the immunogenicity of the insect species with poor immunogenicity is enhanced by an adjuvant. Cytokines such as interleukin-4 (IL-4) have a significant effect on the immunogenicity of the antigen and play a key role in the immune response process. IL-4 is a cytokine secreted by type II helper T-cells (Th2 cells). Its biological effects are mainly to stimulate activated B-cell and T-cell proliferation, and induce the differentiation of immature auxiliary T-cells into type II helper T-cells. It is not clear whether IL-4 has the function of increasing the immunogenicity of coccidia. The effects of the adjuvant effect of ChIL-4 and the effect of ChIL-4 on the infection of coccidiosis were studied in this study. First, we constructed the expression of IL-4 and the expression of the YFP-ethylamine-resistant gene of the Eimeria vector by using the double-expression frame vector strategy. one part of the transfected sub-spore is inoculated to the primary cell of the chicken kidney, and the in-vitro transient transfection efficiency is observed under the fluorescence microscope; the other part is stably transfected and screened under the pressure of the drug under the drug pressure to collect the eggs discharged from 5 to 10 days after the infection, 7 times of successful passage, stable transgenic and Eimeria tenella strains were obtained. In order to compare the level of immune response and the immune protection of the expression of the chicken IL-4 and the wild and slow Eimeria, we divide the 12-day-old AA broiler into three groups, namely, the PBS group, the wild and slow Eimeria, The transgenic and slow Eimeria tenella groups were respectively inoculated with PBS suspension, sporidified wild and slow Eimeria oocysts, sporidified transgenic and slow Eimeria oocysts, and then challenged with two immunizations. The immunogenicity of the two insect strains was compared by the method of measuring the discharge amount of the egg sac, the specific IgY (IgG) antibody drop degree, and the like. The results of the determination of the discharge of the oocysts showed that the number of oocysts was reduced by 6-fold and the peak of the ovulatory capsule was delayed by 6-fold compared with that of the wild and slow Eimeria. the chickens immunized with the transgenic insect strain have better immune protection than the immunized chickens of the wild strain. The results showed that the specific IgY antibody drop in the transgenic rice serum was significantly higher than that of the wild group, indicating that the EChIL-4 stimulated the body to produce a higher humoral immune response than the wild and slow Eimeria. In addition, we compared the egg-sac yield of EmChIL-4 and the wild-parent strain and the effect of the conventional index on the blood of infected chickens. The results showed that infection with EmChIL-4I The egg sac yield of the chicken is significantly lower than that of the chicken infected with the wild mother strain, and the total number of white blood cells, including eosinophils, basophils and lymphocytes, is significantly lower than that of the chicken infected with the wild mother strain; It is suggested that the expression of ChIL-4 in the transgenic and mild Eimeria tenella is weak and the Eimeria tenella is weak. In conclusion, the expression of the chicken IL-4 transgenic and the slow Eimeria tenella has obtained higher immunogenicity and lower virulence, suggesting that the transgene and the slow Eimeria tenella expressing the cytokine IL-4 have the potential as a component of the high-efficiency anti-coccidiosis vaccine, to help the chicken group to establish a better immune protection against the coccidian infection.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.31

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