表達(dá)雞IL-4轉(zhuǎn)基因和緩艾美耳球蟲(chóng)的構(gòu)建及其繁殖力、免疫原性和對(duì)雞血液白細(xì)胞數(shù)量的影響
發(fā)布時(shí)間：2019-01-10 10:21
【摘要】：雞球蟲(chóng)病是嚴(yán)重危害養(yǎng)雞業(yè)的一種寄生原蟲(chóng)病,由頂復(fù)門(mén)艾美耳屬球蟲(chóng)感染引起,每年給世界養(yǎng)雞業(yè)造成愈20億美元的經(jīng)濟(jì)損失。強(qiáng)毒與弱毒球蟲(chóng)病活疫苗被認(rèn)為是預(yù)防禽球蟲(chóng)病的最有效方法之一。盡管可通過(guò)球蟲(chóng)病活疫苗控制雞球蟲(chóng)病的發(fā)生,但由于有些球蟲(chóng)種的免疫原性較差,只能使宿主產(chǎn)生部分免疫保護(hù),致使這些球蟲(chóng)種多次反復(fù)感染而負(fù)面影響了免疫雞群的飼料報(bào)酬,使得球蟲(chóng)病疫苗在肉雞上的使用受到了限制。因此,為了更有效的預(yù)防雞球蟲(chóng)病,通過(guò)佐劑增強(qiáng)免疫原性較差的蟲(chóng)種的免疫原性變得十分重要。細(xì)胞因子如白細(xì)胞介素-4(IL-4),對(duì)抗原的免疫原性有顯著影響,在免疫應(yīng)答過(guò)程中扮演著關(guān)鍵角色。IL-4是Ⅱ型輔助T細(xì)胞(Th2細(xì)胞)分泌的細(xì)胞因子,其生物學(xué)作用主要為刺激活化的B細(xì)胞和T細(xì)胞增殖,誘導(dǎo)未成熟的輔助T細(xì)胞分化成Ⅱ型輔助T細(xì)胞。IL-4是否有提高球蟲(chóng)免疫原性的功能尚不清楚。本研究構(gòu)建了一株表達(dá)雞白細(xì)胞介素-4(ChIL-4)的轉(zhuǎn)基因和緩艾美耳球蟲(chóng)(EmiChIL-4),研究ChIL-4的佐劑效應(yīng)及其在抗球蟲(chóng)感染中的作用。首先,我們利用雙表達(dá)框載體策略構(gòu)建了表達(dá)白細(xì)胞介素-4以及表達(dá)黃熒光蛋白(YFP)-乙胺嘧啶抗性基因的球蟲(chóng)轉(zhuǎn)染載體,將其以核轉(zhuǎn)染法轉(zhuǎn)染和緩艾美耳球蟲(chóng)子孢子。轉(zhuǎn)染的子孢子一部分接種至雞腎原代細(xì)胞,在熒光顯微鏡下觀察體外瞬時(shí)轉(zhuǎn)染效率；另一部分經(jīng)泄殖腔接種雞進(jìn)行藥物壓力下穩(wěn)定轉(zhuǎn)染篩選,收集感染后5-10天排出的卵囊,孢子化后進(jìn)行傳代,成功傳代7次,獲得了穩(wěn)定的轉(zhuǎn)基因和緩艾美耳球蟲(chóng)蟲(chóng)株。為了比較表達(dá)雞IL-4轉(zhuǎn)基因和緩艾美耳球蟲(chóng)(EmChlL-4)和野生和緩艾美耳球蟲(chóng)(wild type)激發(fā)機(jī)體產(chǎn)生的免疫應(yīng)答水平和免疫保護(hù)力,我們將12只12日齡的AA肉雞分成三組,即PBS組、野生和緩艾美耳球蟲(chóng)組、轉(zhuǎn)基因和緩艾美耳球蟲(chóng)組,分別口服接種PBS懸液、孢子化野生和緩艾美耳球蟲(chóng)卵囊、孢子化轉(zhuǎn)基因和緩艾美耳球蟲(chóng)卵囊,兩次免疫后進(jìn)行攻毒。通過(guò)測(cè)定卵囊排出量、特異性IgY (IgG)抗體滴度等實(shí)驗(yàn)方法來(lái)比較兩種蟲(chóng)株的免疫原性。卵囊排出量測(cè)定結(jié)果顯示：相比野生和緩艾美耳球蟲(chóng)組,轉(zhuǎn)基因和緩艾美耳球蟲(chóng)組的卵囊排出量減少了6倍,排卵囊高峰期延遲了22小時(shí),表明轉(zhuǎn)基因蟲(chóng)株免疫的雞比野生株免疫的雞建立了更好的免疫保護(hù)。ELISA結(jié)果顯示轉(zhuǎn)基因組血清中特異性IgY抗體滴度明顯高于野生組,表明了相比野生和緩艾美耳球蟲(chóng),EmChIL-4激發(fā)機(jī)體產(chǎn)生了更高的體液免疫應(yīng)答。另外,我們還比較了EmChIL-4與野生母株的卵囊產(chǎn)量及對(duì)感染雞血液常規(guī)指數(shù)的影響。結(jié)果顯示感染EmChIL-4 I'的雞的卵囊產(chǎn)量顯著低于感染野生母株的雞的卵囊產(chǎn)量；且白細(xì)胞總數(shù),包括嗜酸性粒細(xì)胞、嗜堿性粒細(xì)胞和淋巴細(xì)胞與感染野生母株的雞相比明顯降低；提示ChIL-4在轉(zhuǎn)基因和緩艾美耳球蟲(chóng)中的表達(dá)致弱了和緩艾美耳球蟲(chóng)。綜上所述,表達(dá)雞IL-4轉(zhuǎn)基因和緩艾美耳球蟲(chóng)獲得了更高的免疫原性和更低的毒力,提示表達(dá)細(xì)胞因子IL-4的轉(zhuǎn)基因和緩艾美耳球蟲(chóng)有作為高效抗球蟲(chóng)病疫苗的組成成分的潛力,幫助雞群建立對(duì)球蟲(chóng)感染更好的免疫保護(hù)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.31
本文編號(hào)：2406203
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.31
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