分子佐剂通过粘膜免疫途径增强草原兔尾鼠卵透明带3DNA疫苗抗生育效果的研究

发布时间：2018-01-14 08:10

本文关键词：分子佐剂通过粘膜免疫途径增强草原兔尾鼠卵透明带3DNA疫苗抗生育效果的研究　出处：《新疆大学》2011年博士论文　论文类型：学位论文

【摘要】：草原兔尾鼠(Lagurus lagurus)属啮齿目仓鼠科田鼠亚科兔尾鼠属,是新疆荒漠草场的一种野生害鼠,对新疆荒漠草场造成巨大的损害,严重威胁着新疆畜牧业的发展。化学毒杀、诱捕、射杀等传统方法时效短、物种专一性差,对环境造成破坏,不能有效控制害鼠的种群数量。采用免疫不育技术降低有害动物的生育率是一种更人道的、物种专一性更强的防治有害动物的方法。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发育过程中发挥重要作用的蛋白或激素为抗原。肌肉和皮下接种等免疫不育疫苗传统接种方式很难在野外施用以控制有害动物种群数量,而口服饵料等粘膜免疫方式可以对有害动物进行大量接种,是切实可行的方法。鼠卵透明带3(Zona pellucida 3)主要由3种糖蛋白(ZP1、ZP2、ZP3)组成, ZP3作为精子的初级受体,诱发顶体反应,其抗体可以阻止精卵结合,在生殖过程中起重要作用,因此成为免疫不育疫苗的理想靶抗原。 DNA疫苗既能激发体液免疫,又能诱导细胞免疫,且具有制备简单、易于贮存运输等诸多优点,已被广泛地应用于免疫不育疫苗的研究中。由于DNA疫苗存在免疫原性不强,免疫效果不佳等原因,因此需要通过添加佐剂,改变发送途径,优化载体等方式来提高DNA不育疫苗的抗生育效果。为了筛选出能有效提高LZP3DNA不育疫苗抗生育效果的分子佐剂,本研究选用小鼠白细胞介素31、白细胞介素15、白细胞介素33和非细胞因子FAI3作为分子佐剂,通过粘膜免疫途径,增强草原兔尾鼠LZP3DNA不育疫苗抗生育效果。并进一步探讨细胞因子和非细胞因子FAI3作为佐剂对草原兔尾鼠卵透明带DNA不育疫苗的免疫调节作用。白细胞介素31主要由活化的Th2细胞生成,在T细胞介导的免疫反应中起重要作用,它参与了炎症和变性皮肤疾病。在遗传过敏性皮炎和接触性过敏性皮炎患者中,IL-31表达水平的增高与IL-4和IL-13具有一定的相关性。细胞因子白细胞介素33(interleukin 33,IL-33)是IL-1家族的新成员。IL-33通过IL-1受体ST2发挥其生物学功能,活化NF-kB和MAP激酶,在体外促进TH2细胞产生TH2相关细胞因子。人IL-33的表达仅限于支气管、小气道的上皮细胞、成纤维细胞以及平滑肌细胞,这暗示IL-33有可能参与了黏膜免疫。DC细胞通过ST2直接对IL-33产生应答,被IL-33活化的DC细胞触发了一种非典型的Th2型免疫反应,生成IL-5和IL-13。IL-33与DC细胞的相互作用可能代表了一种引发Th2型免疫反应的新途径。白细胞介素15是一个促炎症细胞因子,通过诱导淋巴细胞的活化、增殖,细胞因子的释放来增强体液和细胞免疫反应。IL-15充当佐剂时,能提高抗体滴度,促进DC细胞成熟。 FAI(Fbrinogen/ albumin/ IgG,纤维蛋白原/白蛋白/免疫球蛋白G受体)是来自C群链球菌的一种多配基结合蛋白,它能同时结合纤维蛋白原、白蛋白和免疫球蛋白G。位于415bp-702bp处的fai3基因片段具有粘膜佐剂活性。首先克隆获得了小鼠细胞因子白细胞介素33基因、白细胞介素31基因和白细胞介素31突变体。成功构建了pcD- mIL-15、pcD- mIL31、pcD- muIL31、pcD- mIL33和pcD- fai3五种重组质粒,这五种质粒均能在真核细胞中表达相应的佐剂分子。用壳聚糖chitosan作为发送载体,chitosan同时或单独包裹DNA疫苗pcD-Lzp3和重组质粒pcD-mIL-15、pcD-mIL31、pcD-muIL31、pcD-mIL33和pcD- fai3形成不同的chitosan-DNA复合物。在第0, 14, 28和42d,分别用不同的chitosan-DNA复合物通过滴鼻免疫途径免疫ICR小鼠。间接ELISA检测血清中抗LZP3特异性IgG、IgG1和IgG2a抗体,以及阴道洗液和粪便中的特异性IgA抗体。结果显示:滴鼻共免疫chi-(pcD-Lzp3+pcD-mIL-31)或chi-(pcD-Lzp3+pcD-muIL-31)都能诱导机体产生较高水平的血清IgG,降低了雌鼠的平均窝仔数。这两个共免疫组都产生较强的淋巴细胞增殖活性,激发Th1型免疫反应。共免疫chi-(pcD-Lzp3+pcD- muIL-31)不育小鼠的卵巢形态异常;在chi-(pcD-Lzp3+ pcD-mIL-31)组,不育小鼠卵巢中卵泡数量明显减少。 Chi-(pcD-Lzp3+pcD-mIL-15)产生较强的淋巴细胞增殖活性。chi-(pcD-Lzp3+pcD-mIL-15)共免疫组小鼠的卵巢组织具有各级形态发育正常的卵泡,无卵巢炎的发生。滴鼻共免疫chi-(pcD-Lzp3+pcD-mIL-33)诱导产生了最高水平的血清IgG和粘膜sIgA,生育率和平均窝仔数显著降低。Chi-(pcD-Lzp3+pcD-mIL-33)共免疫组不育小鼠的卵巢组织出现了卵泡萎缩、卵母细胞丢失等异常现象。这些研究结果表明, mIL-33作为分子佐剂与DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,能显著提高DNA不育疫苗抗生育效果,并能够诱导机体产生较强的系统体液免疫反应和粘膜免疫反应。 Chi-(pcD-Lzp3+pcD-fai3)滴鼻共免疫也诱导机体生成了抗Lzp3特异性血清IgG和黏膜sIgA,生育率和平均窝仔数显著降低。共免疫组小鼠的卵巢组织具有各级形态发育正常的卵泡,无卵巢炎的发生。结果显示,fai3作为分子佐剂与DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠增强了体液免疫反应和黏膜免疫反应,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)共免疫组,免疫接种后不育小鼠的抗体水平显著高于生育小鼠,小鼠不育与抗体水平之间具有明显的相关性。口服免疫是不育疫苗发送的有效途径。本研究进一步探讨了小鼠白细胞介素15和FAI3作为口服粘膜佐剂增强Lzp3 DNA不育疫苗抗生育效果的可行性。将上述chitosan-DNA复合物加入鼠粮中,制成饵料,免疫ICR小鼠。结果显示,口服共免疫chi-(pcD-Lzp3+pcD-fai3)都诱导产生了较高水平的血清IgG和肠粘膜sIgA,生育率和平均窝仔数都有所下降。Chi-(pcD-Lzp3+pcD-mIL-15)免疫组产生了较强的淋巴细胞增殖活性。对卵巢切片进行组织学检测,结果显示,两个共免疫组小鼠的卵巢组织正常。综上所述,小鼠细胞因子白细胞介素33作为分子佐剂与草原兔尾鼠DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,增强了机体的系统体液免疫反应和粘膜免疫反应,提高了DNA不育疫苗的抗生育率。白细胞介素31、白细胞介素15与草原兔尾鼠DNA不育疫苗pcD-Lzp3滴鼻共免疫小鼠,所产生的抗体水平和抗生育率均低于chi-(pcD-Lzp3+pcD-mIL-33)共免疫组。这些结果说明小鼠细胞因子白细胞介素33与白细胞介素31、白细胞介素15相比较,具有更强的分子佐剂功效。非细胞因子FAI3作为分子佐剂与DNA不育疫苗pcD-Lzp3,通过滴鼻和口服两种黏膜免疫途径共免疫小鼠,不仅增强了体液免疫反应,还激发了机体的粘膜免疫反应。因此,mIL-33和FAI3都可以作为分子佐剂,与DNA不育疫苗pcD-Lzp3共免疫,能够显著提高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.

【学位授予单位】：新疆大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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