本文主要研究内容
作者范琳琳（2019）在《十二指肠贾第虫γ和δ贾第素DNA疫苗免疫保护力评价》一文中研究指出：十二指肠贾第虫(Giardia duodenalis,简称贾第虫)是一种常见的厌氧原生动物病原体,广泛引起多种哺乳动物产生胃肠道疾病。贾第虫能够导致腹泻、胀气、消瘦、大便油腻恶臭等症状,并且可能引发慢性疾病和肠道外并发症。随着现代生物技术的不断的创新发展,贾第虫的基因疫苗(DNA疫苗)作为一种可能应用于防治贾第虫病的重要方式,其相关研究也受到了越来越多人的关注。DNA疫苗目前在动物模型上能够成功地预防和治疗各种疾病,具有稳定,方便和安全的特点,并且能够引发机体产生细胞和体液免疫应答反应,能够完善婴儿的抗体应答。pcDNA3.1质粒是一种哺乳动物细胞表达载体,在真核表达载体构建中已被广泛应用,适用于DNA疫苗的开发。CpG序列可诱导强烈的Th1样应答,可作为疫苗的佐剂,抵抗各种目标,包括感染性物质,肿瘤抗原,过敏原等。贾第素(giardin)是组成贾第虫细胞骨架蛋白的重要元素,目前确定的贾第素为α、β、γ和δ-giardin。有关研究表明α和β-giardin分子均具有较高的免疫原性,γ-giardin为38 kDa的蛋白质,也许是微丝带的成分之一。δ-giardin在分子水平上的研究取得了进展,但目前关于它的具体定位还是未知的。α1-giardin是研制贾第虫病疫苗的有效蛋白,这为我们的对贾第素家族的另外两种重要成员γ和δ-giardin的DNA疫苗研究提供了依据。本研究旨在构建贾第虫γ和δ-giardin基因的DNA疫苗,评价其抗贾第虫的免疫保护力。因此,本研究通过RT-PCR扩增得到了截短的γ和δ-giardin基因,将得到的γ和δ-giardin基因连接到真核表达载体pcDNA3.1,并且成功构建了真核表达质粒pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin,进而将他们转染到Vero细胞内并利用间接免疫荧光和Western Blot技术对重组质粒体外表达进行鉴定,然后将真核表达重组质粒肌肉注射入BALB/c小鼠体内进行三次接种免疫,将接种pcDNA3.1空载体的小鼠作为对照组。分别于三次免疫之后采集小鼠血清,利用ELISA技术检测血清中的IgG及其亚型IgG1、IgG2a的抗体水平。第三次免疫之后,利用CCK-8进行脾淋巴细胞增殖实验,并用ELISA技术检测经贾第虫可溶性抗原刺激脾淋巴细胞而产生的细胞因子IFN-γ和IL-4,利用流式细胞术检测脾淋巴细胞中的CD3~+CD4~+CD8~-和CD3~+CD8~+CD4~-T细胞百分比含量。利用贾第虫包囊接种小鼠,对接种后16 d的粪便进行包囊计数。结果显示,pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin成功转染至Vero细胞中并成功表达。接种了三种真核表达质粒的小鼠产生了特异性的体液和细胞反应,与对照组相比IgG和IgG2a抗体水平显著提高。pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin免疫组的SI值与对照组相比有显著的提升,分别为1.09±0.022,1.07±0.021,1.05±0.023。CD3+CD4+CD8-和CD3+CD8+CD4-T细胞百分比显著提高,CD4~+T细胞百分比分别为23.65±0.78,23.20±0.99,21.25±0.64;CD8~+T细胞百分比分别为10.90±0.99,11.80±1.27,9.90±0.57。接种了pcDNA3.1-γ-giardin和pcDNA3.1-γ-δ-giardin的细胞因子IL-4和IFN-γ显著提高,接种pcDNA3.1-δ-giardin免疫组的IFN-γ水平显著提高。攻虫实验结果显示,接种pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin基因疫苗的小鼠的减虫率分别为39.1%、26.1%和18.3%。本实验成功构建了真核重组表达载体pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin,并评价其引起小鼠产生的免疫保护效果。上述结果表明pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardin和pcDNA3.1-γ-δ-giardin能够诱导小鼠产生一定水平的体液免疫应答和细胞免疫应答,并在机体对抗贾第虫感染时产生一定的免疫保护力。该研究结果为进一步研发贾第虫DNA疫苗提供了理论基础。
Abstract
shi er zhi chang gu di chong (Giardia duodenalis,jian chen gu di chong )shi yi chong chang jian de ya yang yuan sheng dong wu bing yuan ti ,an fan yin qi duo chong bu ru dong wu chan sheng wei chang dao ji bing 。gu di chong neng gou dao zhi fu xie 、zhang qi 、xiao shou 、da bian you ni e chou deng zheng zhuang ,bing ju ke neng yin fa man xing ji bing he chang dao wai bing fa zheng 。sui zhao xian dai sheng wu ji shu de bu duan de chuang xin fa zhan ,gu di chong de ji yin yi miao (DNAyi miao )zuo wei yi chong ke neng ying yong yu fang zhi gu di chong bing de chong yao fang shi ,ji xiang guan yan jiu ye shou dao le yue lai yue duo ren de guan zhu 。DNAyi miao mu qian zai dong wu mo xing shang neng gou cheng gong de yu fang he zhi liao ge chong ji bing ,ju you wen ding ,fang bian he an quan de te dian ,bing ju neng gou yin fa ji ti chan sheng xi bao he ti ye mian yi ying da fan ying ,neng gou wan shan ying er de kang ti ying da 。pcDNA3.1zhi li shi yi chong bu ru dong wu xi bao biao da zai ti ,zai zhen he biao da zai ti gou jian zhong yi bei an fan ying yong ,kuo yong yu DNAyi miao de kai fa 。CpGxu lie ke you dao jiang lie de Th1yang ying da ,ke zuo wei yi miao de zuo ji ,di kang ge chong mu biao ,bao gua gan ran xing wu zhi ,zhong liu kang yuan ,guo min yuan deng 。gu di su (giardin)shi zu cheng gu di chong xi bao gu jia dan bai de chong yao yuan su ,mu qian que ding de gu di su wei α、β、γhe δ-giardin。you guan yan jiu biao ming αhe β-giardinfen zi jun ju you jiao gao de mian yi yuan xing ,γ-giardinwei 38 kDade dan bai zhi ,ye hu shi wei si dai de cheng fen zhi yi 。δ-giardinzai fen zi shui ping shang de yan jiu qu de le jin zhan ,dan mu qian guan yu ta de ju ti ding wei hai shi wei zhi de 。α1-giardinshi yan zhi gu di chong bing yi miao de you xiao dan bai ,zhe wei wo men de dui gu di su jia zu de ling wai liang chong chong yao cheng yuan γhe δ-giardinde DNAyi miao yan jiu di gong le yi ju 。ben yan jiu zhi zai gou jian gu di chong γhe δ-giardinji yin de DNAyi miao ,ping jia ji kang gu di chong de mian yi bao hu li 。yin ci ,ben yan jiu tong guo RT-PCRkuo zeng de dao le jie duan de γhe δ-giardinji yin ,jiang de dao de γhe δ-giardinji yin lian jie dao zhen he biao da zai ti pcDNA3.1,bing ju cheng gong gou jian le zhen he biao da zhi li pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardin,jin er jiang ta men zhuai ran dao Veroxi bao nei bing li yong jian jie mian yi ying guang he Western Blotji shu dui chong zu zhi li ti wai biao da jin hang jian ding ,ran hou jiang zhen he biao da chong zu zhi li ji rou zhu she ru BALB/cxiao shu ti nei jin hang san ci jie chong mian yi ,jiang jie chong pcDNA3.1kong zai ti de xiao shu zuo wei dui zhao zu 。fen bie yu san ci mian yi zhi hou cai ji xiao shu xie qing ,li yong ELISAji shu jian ce xie qing zhong de IgGji ji ya xing IgG1、IgG2ade kang ti shui ping 。di san ci mian yi zhi hou ,li yong CCK-8jin hang pi lin ba xi bao zeng shi shi yan ,bing yong ELISAji shu jian ce jing gu di chong ke rong xing kang yuan ci ji pi lin ba xi bao er chan sheng de xi bao yin zi IFN-γhe IL-4,li yong liu shi xi bao shu jian ce pi lin ba xi bao zhong de CD3~+CD4~+CD8~-he CD3~+CD8~+CD4~-Txi bao bai fen bi han liang 。li yong gu di chong bao nang jie chong xiao shu ,dui jie chong hou 16 dde fen bian jin hang bao nang ji shu 。jie guo xian shi ,pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardincheng gong zhuai ran zhi Veroxi bao zhong bing cheng gong biao da 。jie chong le san chong zhen he biao da zhi li de xiao shu chan sheng le te yi xing de ti ye he xi bao fan ying ,yu dui zhao zu xiang bi IgGhe IgG2akang ti shui ping xian zhe di gao 。pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardinmian yi zu de SIzhi yu dui zhao zu xiang bi you xian zhe de di sheng ,fen bie wei 1.09±0.022,1.07±0.021,1.05±0.023。CD3+CD4+CD8-he CD3+CD8+CD4-Txi bao bai fen bi xian zhe di gao ,CD4~+Txi bao bai fen bi fen bie wei 23.65±0.78,23.20±0.99,21.25±0.64;CD8~+Txi bao bai fen bi fen bie wei 10.90±0.99,11.80±1.27,9.90±0.57。jie chong le pcDNA3.1-γ-giardinhe pcDNA3.1-γ-δ-giardinde xi bao yin zi IL-4he IFN-γxian zhe di gao ,jie chong pcDNA3.1-δ-giardinmian yi zu de IFN-γshui ping xian zhe di gao 。gong chong shi yan jie guo xian shi ,jie chong pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardinji yin yi miao de xiao shu de jian chong lv fen bie wei 39.1%、26.1%he 18.3%。ben shi yan cheng gong gou jian le zhen he chong zu biao da zai ti pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardin,bing ping jia ji yin qi xiao shu chan sheng de mian yi bao hu xiao guo 。shang shu jie guo biao ming pcDNA3.1-γ-giardin、pcDNA3.1-δ-giardinhe pcDNA3.1-γ-δ-giardinneng gou you dao xiao shu chan sheng yi ding shui ping de ti ye mian yi ying da he xi bao mian yi ying da ,bing zai ji ti dui kang gu di chong gan ran shi chan sheng yi ding de mian yi bao hu li 。gai yan jiu jie guo wei jin yi bu yan fa gu di chong DNAyi miao di gong le li lun ji chu 。
论文参考文献
论文详细介绍
论文作者分别是来自东北农业大学的范琳琳，发表于刊物东北农业大学2019-08-27论文，是一篇关于十二指肠贾第虫论文,贾第素论文,贾第素论文,疫苗论文,免疫保护力论文,东北农业大学2019-08-27论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自东北农业大学2019-08-27论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：十二指肠贾第虫论文; 贾第素论文; 疫苗论文; 免疫保护力论文; 东北农业大学2019-08-27论文;