CN106177956B - 提高鸡抗ndv病毒效果的方法 - Google Patents
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Abstract
提高鸡抗NDV病毒效果的方法，它涉及一种提高禽类抗NDV病毒效果的方法。本发明利用禽先天性免疫系统，通过人为干预，调节抗NDV病毒信号转导，增加鸡体内抗NDV感染的禽β‑防御素分泌量，进而提高鸡对NDV的抗病毒效果。本发明方法利用Toll样受体激动剂刺激鸡体内Toll样受体15(TLR15)、激活p38 MAPK信号转导通路，从而增加鸡体内AvBD2的分泌量，实现抗NDV效果的提高。
Description
技术领域
本发明涉及一种提高禽类抗NDV病毒效果的方法。
背景技术
新城疫(Newcastle Diseases，ND)，俗称亚洲鸡瘟、伪鸡瘟，是由新城疫病毒(Newcastle diseases virus，NDV)强毒引起的主要侵害以鸡为主的多种禽类的一种急性、高度接触性的烈性传染病。
宿主防御肽(Host defensin peptides，HDPs)是生物体产生的一类具有广谱抗菌活性的小分子多肽，是机体抗病原入侵的第一道防线。但在禽类体内仅发现存在β-防御素，即禽β-防御素(avianβ-defensins,AvBDs)。由于不同AvBDs抗病毒作用的信号转导机制不完全一致，所以目前还无法利用禽先天性免疫系统对抗新城疫病毒。
发明内容
本发明利用禽先天性免疫系统，通过人为干预，调节抗NDV病毒信号转导，增加鸡体内抗NDV感染的禽β-防御素分泌量，进而提高鸡对NDV的抗病毒效果。
本发明提高鸡抗NDV病毒效果的方法利用激动剂刺激鸡体内TLR15、激活p38MAPK信号转导通路，从而增加鸡体内AvBD2的分泌量，实现抗NDV效果的提高。
本发明方法利用禽类天然免疫调控机制，提高禽机体的免疫能力，并且避免了耐药性的发生。与使用抗生素的方法相比，应用本发明方法的鸡具有更高的食用安全性。
鸡处于鸡胚阶段，组织和器官尚未发育成熟，此时并不能调动各自的免疫体系，发挥抗NDV作用。鸡胚成纤维细胞发育成熟最早，且数量大。本发明方法可以增加鸡胚成纤维细胞的AvBD2分泌量，实现了鸡胚阶段的抗NDV。
本发明方法
具体实施方式
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。
具体实施方式一：本实施方式提高鸡抗NDV病毒效果的方法利用激动剂刺激鸡体内TLR15、激活p38 MAPK信号转导通路，从而增加鸡体内AvBD2的分泌量，实现抗NDV效果的提高。
具体实施方式二：本实施方式与具体实施方式一的不同点是：注射ODN-M362、Pam3CSK4或FLA-ST刺激TLR15。其它步骤及参数与实施方式一相同。
具体实施方式三：本实施方式与具体实施方式一或二的不同点是：注射积雪草酸(Asiatic acid)。其它步骤及参数与实施方式一或二相同。
实施例
实验1：采用构建PCAG-his-AVBD2、PCAG-AVBD2-his(Xho I/Bam HI酶切位点)真核表达质粒，用Expi293F悬浮培养表达系统表达AVBD2蛋白，并用镍柱纯化后，进一步将不同浓度的AVBD2与NDV(F48E9毒株)相互作用1h后，接种DF-1细胞，分别于36h和48h后，用MTT试剂盒检测细胞凋亡。
实验1结果：表明AVBD2具有抗NDV作用。
真核细胞表达系统，完全模拟体内环境，通过真核细胞的转录加工，对表达的mRNA做剪切修饰，从而使表达的蛋白具有接近体内真实的构象和生物活性。Expi293F真核表达系统可以获得大量高纯度和具有生物活性的目的蛋白。因此制备真核表达质粒在DF-1细胞中进行试验更为接近活鸡防疫实际情况。同时在DF-1细胞中进行试验也避免了活鸡体内其他组织系统的干扰，能够真实地反映出AVBD2对于NDV的抗病毒效果。
实验2：选9日龄SPF级鸡胚制作CEF细胞(鸡胚成纤维细胞)，铺12孔板，12h后去掉培养基，PBS洗涤2次，加入无血清DMEM稀释的NDV(F48E9毒株)，按1MOI(multiplicity ofinfection)比例感染，对照组为无NDV(F48E9毒株)的无血清DMEM，37℃吸附1.5h后，PBS洗涤细胞3次，加入含2％胎牛血清的DMEM培养基在37℃、5％CO2培养箱中继续培养6、12、24、36和48h后弃掉上清，PBS洗3遍细胞后，加入1ml TRIzol提取RNA，应用荧光定量PCR方法检测AvBDs(1～14)的基因表达量。每个时间点设置3～4个平行样本。
实验2结果：经NDV(F48E9毒株)感染后与对照组相比，仅AvBD2的基因表达量显著增加p＜0.05，而其他AvBDs经攻毒后表达量变化不明显。
鸡胚成纤维细胞为鸡胚(成纤维组织)细胞，不含器官和(功能性)组织。
实验3：分别用TLRs激动剂LPS(TLR4)、ODN-M362(TLR21)、Pam3CSK4(TLR1/2)、polyI:C(TLR3)、R848(TLR7)、FLA-ST(TLR5)作用于鸡CEF细胞，各激动剂的用量分别为LPS-B5——2μg/ml，ODN-M362——2.5μM，Pam3CSK4——0.4μg/ml，polyI:C——50μg/ml，R848——5μg/ml，FLA-ST——2μg/ml，对照组为只加相应剂量的H2O。将CEF细胞于37℃、5％CO2培养箱中继续培养，并分别在培养6h、24h和48h后，弃掉培养基，PBS洗涤细胞3次，每个细胞培养孔加入1ml TRIzol提取RNA，应用荧光定量PCR方法检测TLR15和AvBD2的基因表达量。每个时间点设置3～4个平行样本。
实验3结果：TLR15和AvBD2基因表达水平在ODN-M362、Pam3CSK4和FLA-ST刺激后表达量同步显著上调，与未加激动剂的对照组比AvBD2基因表达量增加了10～30倍。
实验4：选9日龄SPF级鸡胚制作CEF铺12孔板，12h后去掉培养基，PBS洗涤2次，加入1ml含2％胎牛血清的DMED培养基，分别加入MAPK(JNK、ERK和p38)和NF-κB通路的抑制剂SP600125(50μM)、PD 98059(50μM)、SB 203580(50μM)和PDTC(50μM)，对照组为加入相应剂量的DMSO，在37℃、5％CO2培养箱中继续培养1h后，分别加入相应浓度的TLRs激动剂(ODN-M362 2.5μM、Pam3CSK4 0.4μg/ml、FLA-ST 2μg/ml)，继续培养箱中培养24h，弃掉细胞上清，PBS洗涤细胞3次，加入1ml TRIzol提取RNA，应用荧光定量PCR方法检测AvBD2的基因表达量。每个时间点设置3～4个平行样本。
实验4结果：仅有SB203580对AvBD2基因的表达量产生显著的抑制作用。说明AvBD2主要通过p38MAPK通路介导产生。
实验5：选9日龄SPF级鸡胚制作CEF铺12孔板，12h后去掉培养基，PBS洗涤2次，加入1ml含2％胎牛血清的含NDV(F48E9毒株)DMED培养基，接毒量为1MOI，分成3组，第1组只加入p38MAPK通路抑制剂SB 203580(50μM)，第2组只加入p38MAPK通路激动剂Asiatic acid(50μM)和抑制剂SB 203580(50μM)，第3组为对照组只加相应量的DMSO或水。在37℃、5％CO2培养箱中继续培养24h，弃掉细胞上清，PBS洗涤细胞3次，加入1ml TRIzol提取RNA，应用荧光定量PCR方法检测AvBD2的基因表达量。每个时间点设置3～4个平行样本。
实验5结果：第2组和第3组AvBD2基因表达水平基本相同。证明Asiatic acid能够抵消p38MAPK通路抑制剂的作用，激活p38MAPK信号转导通路，增加AvBD2基因表达量。
Claims (2)
1.刺激鸡体内TLR15的激动剂在制备增加鸡体内AvBD2分泌量的药物中的应用，其特征在于刺激鸡体内TLR15的激动剂作为增加鸡体内AvBD2分泌量药物的活性成分；其中所述的激动剂为ODN-M362、Pam3CSK4或FLA-ST。
2.刺激鸡体内p38 MAPK通路的激动剂在制备增加鸡体内AvBD2分泌量的药物中的应用，其特征在于刺激鸡体内p38 MAPK通路的激动剂作为增加鸡体内AvBD2分泌量药物的活性成分；其中所述的激动剂为积雪草酸。
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| CN101538582A (zh) * | 2009-04-30 | 2009-09-23 | 东北农业大学 | 天然抗菌剂鸭β-防御素2重组蛋白的制备方法 |
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| "TLRs信号通路和促炎症细胞因子基因在鸡胚成纤维细胞感染新城疫病毒过程中的表达分析";曾胜强等;《四川农业大学学报》;20141231;第32卷(第4期);第436-441页 * |
| "两种不同来源新城疫病毒的感染对鸡免疫作用的分子机制";张婷婷;《中国优秀硕士学位论文全文数据库 农业科技辑》;中国学术期刊（光盘版）电子杂志社;20160415;第D050-552页 * |
| "感染不同毒力新城疫病毒后鸡免疫器官Toll样受体及炎症相关基因的表达差异";程洋;《中国优秀硕士学位论文全文数据库 农业科技辑》;中国学术期刊（光盘版）电子杂志社;20160715;第D050-423页 * |
| "积雪草酸对新生大鼠心肌细胞缺血-再灌注损伤的保护作用";易晨龙等;《江苏医药》;20150831;第41卷(第15期);第1743-1746页 * |
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| Publication number | Publication date |
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| CN106177956A (zh) | 2016-12-07 |
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