本文關(guān)鍵詞：分子佐劑通過粘膜免疫途徑增強草原兔尾鼠卵透明帶3DNA疫苗抗生育效果的研究　出處：《新疆大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 草原兔尾鼠 卵透明帶3 DNA疫苗 分子佐劑 粘膜免疫 抗生育

【摘要】：草原兔尾鼠(Lagurus lagurus)屬嚙齒目倉鼠科田鼠亞科兔尾鼠屬,是新疆荒漠草場的一種野生害鼠,對新疆荒漠草場造成巨大的損害,嚴重威脅著新疆畜牧業(yè)的發(fā)展。化學(xué)毒殺、誘捕、射殺等傳統(tǒng)方法時效短、物種專一性差,對環(huán)境造成破壞,不能有效控制害鼠的種群數(shù)量。采用免疫不育技術(shù)降低有害動物的生育率是一種更人道的、物種專一性更強的防治有害動物的方法。 免疫不育疫苗主要以精子或卵子蛋白以及在受精和胚胎早期發(fā)育過程中發(fā)揮重要作用的蛋白或激素為抗原。肌肉和皮下接種等免疫不育疫苗傳統(tǒng)接種方式很難在野外施用以控制有害動物種群數(shù)量,而口服餌料等粘膜免疫方式可以對有害動物進行大量接種,是切實可行的方法。鼠卵透明帶3(Zona pellucida 3)主要由3種糖蛋白(ZP1、ZP2、ZP3)組成, ZP3作為精子的初級受體,誘發(fā)頂體反應(yīng),其抗體可以阻止精卵結(jié)合,在生殖過程中起重要作用,因此成為免疫不育疫苗的理想靶抗原。 DNA疫苗既能激發(fā)體液免疫,又能誘導(dǎo)細胞免疫,且具有制備簡單、易于貯存運輸?shù)戎T多優(yōu)點,已被廣泛地應(yīng)用于免疫不育疫苗的研究中。由于DNA疫苗存在免疫原性不強,免疫效果不佳等原因,因此需要通過添加佐劑,改變發(fā)送途徑,優(yōu)化載體等方式來提高DNA不育疫苗的抗生育效果。 為了篩選出能有效提高LZP3DNA不育疫苗抗生育效果的分子佐劑,本研究選用小鼠白細胞介素31、白細胞介素15、白細胞介素33和非細胞因子FAI3作為分子佐劑,通過粘膜免疫途徑,增強草原兔尾鼠LZP3DNA不育疫苗抗生育效果。并進一步探討細胞因子和非細胞因子FAI3作為佐劑對草原兔尾鼠卵透明帶DNA不育疫苗的免疫調(diào)節(jié)作用。 白細胞介素31主要由活化的Th2細胞生成,在T細胞介導(dǎo)的免疫反應(yīng)中起重要作用,它參與了炎癥和變性皮膚疾病。在遺傳過敏性皮炎和接觸性過敏性皮炎患者中,IL-31表達水平的增高與IL-4和IL-13具有一定的相關(guān)性。細胞因子白細胞介素33(interleukin 33,IL-33)是IL-1家族的新成員。IL-33通過IL-1受體ST2發(fā)揮其生物學(xué)功能,活化NF-kB和MAP激酶,在體外促進TH2細胞產(chǎn)生TH2相關(guān)細胞因子。人IL-33的表達僅限于支氣管、小氣道的上皮細胞、成纖維細胞以及平滑肌細胞,這暗示IL-33有可能參與了黏膜免疫。DC細胞通過ST2直接對IL-33產(chǎn)生應(yīng)答,被IL-33活化的DC細胞觸發(fā)了一種非典型的Th2型免疫反應(yīng),生成IL-5和IL-13。IL-33與DC細胞的相互作用可能代表了一種引發(fā)Th2型免疫反應(yīng)的新途徑。白細胞介素15是一個促炎癥細胞因子,通過誘導(dǎo)淋巴細胞的活化、增殖,細胞因子的釋放來增強體液和細胞免疫反應(yīng)。IL-15充當佐劑時,能提高抗體滴度,促進DC細胞成熟。 FAI(Fbrinogen/ albumin/ IgG,纖維蛋白原/白蛋白/免疫球蛋白G受體)是來自C群鏈球菌的一種多配基結(jié)合蛋白,它能同時結(jié)合纖維蛋白原、白蛋白和免疫球蛋白G。位于415bp-702bp處的fai3基因片段具有粘膜佐劑活性。 首先克隆獲得了小鼠細胞因子白細胞介素33基因、白細胞介素31基因和白細胞介素31突變體。成功構(gòu)建了pcD- mIL-15、pcD- mIL31、pcD- muIL31、pcD- mIL33和pcD- fai3五種重組質(zhì)粒,這五種質(zhì)粒均能在真核細胞中表達相應(yīng)的佐劑分子。用殼聚糖chitosan作為發(fā)送載體,chitosan同時或單獨包裹DNA疫苗pcD-Lzp3和重組質(zhì)粒pcD-mIL-15、pcD-mIL31、pcD-muIL31、pcD-mIL33和pcD- fai3形成不同的chitosan-DNA復(fù)合物。在第0, 14, 28和42d,分別用不同的chitosan-DNA復(fù)合物通過滴鼻免疫途徑免疫ICR小鼠。間接ELISA檢測血清中抗LZP3特異性IgG、IgG1和IgG2a抗體,以及陰道洗液和糞便中的特異性IgA抗體。 結(jié)果顯示:滴鼻共免疫chi-(pcD-Lzp3+pcD-mIL-31)或chi-(pcD-Lzp3+pcD-muIL-31)都能誘導(dǎo)機體產(chǎn)生較高水平的血清IgG,降低了雌鼠的平均窩仔數(shù)。這兩個共免疫組都產(chǎn)生較強的淋巴細胞增殖活性,激發(fā)Th1型免疫反應(yīng)。共免疫chi-(pcD-Lzp3+pcD- muIL-31)不育小鼠的卵巢形態(tài)異常;在chi-(pcD-Lzp3+ pcD-mIL-31)組,不育小鼠卵巢中卵泡數(shù)量明顯減少。 Chi-(pcD-Lzp3+pcD-mIL-15)產(chǎn)生較強的淋巴細胞增殖活性。chi-(pcD-Lzp3+pcD-mIL-15)共免疫組小鼠的卵巢組織具有各級形態(tài)發(fā)育正常的卵泡,無卵巢炎的發(fā)生。滴鼻共免疫chi-(pcD-Lzp3+pcD-mIL-33)誘導(dǎo)產(chǎn)生了最高水平的血清IgG和粘膜sIgA,生育率和平均窩仔數(shù)顯著降低。Chi-(pcD-Lzp3+pcD-mIL-33)共免疫組不育小鼠的卵巢組織出現(xiàn)了卵泡萎縮、卵母細胞丟失等異�，F(xiàn)象。這些研究結(jié)果表明, mIL-33作為分子佐劑與DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,能顯著提高DNA不育疫苗抗生育效果,并能夠誘導(dǎo)機體產(chǎn)生較強的系統(tǒng)體液免疫反應(yīng)和粘膜免疫反應(yīng)。 Chi-(pcD-Lzp3+pcD-fai3)滴鼻共免疫也誘導(dǎo)機體生成了抗Lzp3特異性血清IgG和黏膜sIgA,生育率和平均窩仔數(shù)顯著降低。共免疫組小鼠的卵巢組織具有各級形態(tài)發(fā)育正常的卵泡,無卵巢炎的發(fā)生。結(jié)果顯示,fai3作為分子佐劑與DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠增強了體液免疫反應(yīng)和黏膜免疫反應(yīng),提高了DNA不育疫苗的抗生育效果。在chi-(pcD-Lzp3+pcD-fai3)和chi-(pcD-Lzp3+pcD-mIL-33)共免疫組,免疫接種后不育小鼠的抗體水平顯著高于生育小鼠,小鼠不育與抗體水平之間具有明顯的相關(guān)性。 口服免疫是不育疫苗發(fā)送的有效途徑。本研究進一步探討了小鼠白細胞介素15和FAI3作為口服粘膜佐劑增強Lzp3 DNA不育疫苗抗生育效果的可行性。將上述chitosan-DNA復(fù)合物加入鼠糧中,制成餌料,免疫ICR小鼠。結(jié)果顯示,口服共免疫chi-(pcD-Lzp3+pcD-fai3)都誘導(dǎo)產(chǎn)生了較高水平的血清IgG和腸粘膜sIgA,生育率和平均窩仔數(shù)都有所下降。Chi-(pcD-Lzp3+pcD-mIL-15)免疫組產(chǎn)生了較強的淋巴細胞增殖活性。對卵巢切片進行組織學(xué)檢測,結(jié)果顯示,兩個共免疫組小鼠的卵巢組織正常。 綜上所述,小鼠細胞因子白細胞介素33作為分子佐劑與草原兔尾鼠DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,增強了機體的系統(tǒng)體液免疫反應(yīng)和粘膜免疫反應(yīng),提高了DNA不育疫苗的抗生育率。白細胞介素31、白細胞介素15與草原兔尾鼠DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,所產(chǎn)生的抗體水平和抗生育率均低于chi-(pcD-Lzp3+pcD-mIL-33)共免疫組。這些結(jié)果說明小鼠細胞因子白細胞介素33與白細胞介素31、白細胞介素15相比較,具有更強的分子佐劑功效。非細胞因子FAI3作為分子佐劑與DNA不育疫苗pcD-Lzp3,通過滴鼻和口服兩種黏膜免疫途徑共免疫小鼠,不僅增強了體液免疫反應(yīng),還激發(fā)了機體的粘膜免疫反應(yīng)。因此,mIL-33和FAI3都可以作為分子佐劑,與DNA不育疫苗pcD-Lzp3共免疫,能夠顯著提高DNA不育疫苗pcD-Lzp3的抗生育效果。
[Abstract]:Laguruslagurus (Lagurus Lagurus) belongs to Rodentia Cricetidae Microtinae Lagurus, is a kind of wild rodents in Xinjiang desert grassland, caused great damage to the Xinjiang desert, a serious threat to the development of animal husbandry in Xinjiang. Chemical poisoning, trapping, shooting and other traditional methods of short time, species specific difference, causing damage to the environment, the population can not effectively control mice. The immune infertility technology to reduce the harmful animal fertility is a more humane animal control methods, harmful species specific stronger.
Immunocontraceptive vaccine to sperm or egg protein as well as in the process of fertilization and early embryonic development play an important role in protein or hormone as antigen. Intramuscular and subcutaneous inoculation of immunocontraceptive vaccine inoculation method is very difficult in the field of traditional application to control the amount of harmful animal populations, and bait oral mucosal immune way can be a lot of harmful inoculation animal, is a feasible method. Mouse zona pellucida 3 (Zona pellucida 3) is mainly composed of 3 glycoproteins (ZP1, ZP2, ZP3, ZP3) as the primary sperm receptor, induced acrosome reaction, the antibody can block sperm egg binding, plays an important role in the reproductive process, thus become the ideal target antigen immunocontraceptive vaccine.
DNA vaccine can stimulate humoral immunity, and can induce cellular immunity, and has the advantages of simple preparation, easy storage and transportation of many advantages, has been widely used in immunocontraceptive vaccines. The immunogenicity of DNA vaccine is not strong, cause immune effect is poor, because of the need to change by adding adjuvant. Sent, optimization of carrier and other ways to improve the immune effect of DNA contraceptive vaccine.
In order to molecular adjuvant screening can effectively improve the antifertility effect of LZP3DNA vaccine sterility, this study used murine interleukin 31, interleukin 15, interleukin 33 and non cytokine FAI3 as molecular adjuvant, through the mucosal immune pathway, enhanced laguruslagurus LZP3DNA sterile vaccine anti fertility effect and. Further study of cytokines and cytokine regulation of non FAI3 as adjuvant of laguruslagurus zona pellucida DNA sterile vaccine.
Interleukin 31 is mainly composed of Th2 cells activation, play an important role in the immune response mediated by T cells, it is involved in the inflammatory and degenerative disease of the skin. In patients with atopic dermatitis and allergic contact dermatitis, the expression level of IL-31 increased and correlated with IL-4 and IL-13 cells. Factor interleukin 33 (interleukin 33, IL-33) is a new member of IL-1 family,.IL-33 exerts its biological function through the IL-1 receptor ST2, NF-kB and MAP kinase activation in vitro, promoting the production of TH2 related cytokines in TH2 cells. Expression of IL-33 is limited to the bronchi, small airway epithelial cells, fibroblasts and smooth muscle cells, suggesting that IL-33 may be involved in the mucosal immune cells by.DC ST2 directly to the IL-33 response, DC cells by activation of IL-33 triggering Th2 immune response to an atypical, generation of IL-5 and IL-13.IL The interaction between -33 and DC cells may represent a new way of triggering Th2 type immune response. Interleukin 15 is a proinflammatory cytokine, through activation, induced lymphocyte proliferation, cytokine release to enhance humoral and cellular immune responses of.IL-15 as adjuvant, could enhance the antibody titers to promote the maturation of DC cells.
FAI (Fbrinogen/ albumin/ IgG, fibrinogen / albumin / immunoglobulin G receptor) is a ligand binding protein from Streptococcus group C, it can be combined with the fai3 gene fragment of fibrinogen, albumin and immunoglobulin G. is located at 415bp-702bp with a mucosal adjuvant activity.
The first cloned mouse cytokine interleukin 33 gene, interleukin 31 and interleukin 31 gene mutant had been constructed successfully. PcD- mIL-15, pcD- mIL31, pcD- muIL31, pcD- mIL33 and pcD- fai3 five recombinant plasmids, the five plasmids could express in eukaryotic corresponding molecular adjuvant the use of chitosan chitosan cells. As a transmission carrier, chitosan simultaneously or individually wrapped DNA pcD-Lzp3 vaccine and recombinant plasmid pcD-mIL-15, pcD-mIL31, pcD-muIL31, pcD-mIL33 and pcD- fai3 chitosan-DNA complex formation different. In zeroth, 14, 28 and 42d, respectively chitosan-DNA different complexes by intranasal immunization approach to immune ICR mice. Anti LZP3 specific IgG indirect ELISA detection in serum IgG1 and IgG2a antibodies, and vaginal lotion and feces by specific IgA antibody.
The results showed that intranasal co immunization of chi- (pcD-Lzp3+pcD-mIL-31) or chi- (pcD-Lzp3+pcD-muIL-31) can induce high levels of serum IgG, reduce the average litter size of female rats. The number of the two co immunization group had strong lymphocyte proliferation, stimulate the immune response to Th1. Chi- (pcD-Lzp3+pcD- muIL-31) were immunized abnormal ovarian morphology in infertile mice; chi- (pcD-Lzp3+ pcD-mIL-31) group, the number of follicles in the ovary of infertile mice was significantly reduced.
Chi- (pcD-Lzp3+pcD-mIL-15) have strong.Chi- lymphocyte proliferation activity (pcD-Lzp3+pcD-mIL-15) of ovarian tissue of immunized mice with normal levels of follicle morphogenesis, without ovarian inflammation. Intranasal co immunization of chi- (pcD-Lzp3+pcD-mIL-33) induced the highest level of serum IgG and mucosal sIgA, the fertility rate and the average litter size was significantly decreased.Chi- (pcD-Lzp3+pcD-mIL-33) of ovarian tissue sterile mice co immunized group appeared follicular atrophy, abnormal oocyte loss. These results suggest that mIL-33 as a molecular adjuvant DNA vaccine with sterile pcD-Lzp3 intranasally co immunized mice, can significantly improve the antifertility effect of DNA vaccine sterility, and can induce strong system humoral and mucosal immune responses.
Chi- (pcD-Lzp3+pcD-fai3) were also induced by intranasal immune body to produce anti Lzp3 specific serum IgG and mucosal sIgA, fertility rate and average litter size significantly decreased. The ovarian tissue of immunized mice with normal levels of follicle morphogenesis, without ovarian inflammation. The results showed that fai3 and DNA as molecular adjuvant sterile vaccine pcD-Lzp3 intranasally co immunized mice enhanced the humoral immune responses and mucosal immune response, improve the immune effect of DNA contraceptive vaccine. The chi- (pcD-Lzp3+pcD-fai3) and chi- (pcD-Lzp3+pcD-mIL-33) Co immunized group, antibody level in mice after immunization with male fertility was significantly higher than that in mice, there was a significant correlation between infertility and mice the antibody level.
Oral immunization is an effective way to send the sterility vaccine. This study further discusses the mouse interleukin 15 FAI3 Lzp3 DNA and feasibility of enhancing the anti fertility effect of the sterile vaccine as oral mucosal adjuvant. The chitosan-DNA complex with rat food, made of food, immunization of ICR mice. The results showed that the total oral immune chi- (pcD-Lzp3+pcD-fai3) all induced serum IgG and intestinal mucosal sIgA high level, the fertility rate and average litter size decreased.Chi- (pcD-Lzp3+pcD-mIL-15) immunity group produced strong activity of lymphocyte proliferation. Ovarian sections for histological examination. The results showed that two ovarian tissues of immunized mice.
In summary, mouse cytokine interleukin 33 as molecular adjuvant and laguruslagurus DNA sterile vaccine pcD-Lzp3 intranasally co immunized mice, enhance the systemic and mucosal immune responses, improve the anti fertility DNA sterility vaccine. Interleukin 31, interleukin 15 and grassland Lagurus DNA sterile vaccine pcD-Lzp3 intranasally co immunized mice, the antibody level and anti fertility rate is lower than that of chi- (pcD-Lzp3+pcD-mIL-33) were immunized group. These results suggest that mouse cytokine interleukin 33 and interleukin 31, interleukin 15 compared the molecular adjuvant efficacy has stronger non cytokine FAI3 as molecular adjuvant DNA vaccine and sterile pcD-Lzp3 by intranasal and oral administration of two mucosal immune co immunized mice, not only enhances the humoral immune response, but also to stimulate the mucosal immune reaction Therefore, both mIL-33 and FAI3 can be used as a molecular adjuvant, CO immunized with the DNA sterile vaccine pcD-Lzp3, and can significantly improve the anti fertility effect of the DNA sterile vaccine pcD-Lzp3.

【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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