【作者】 赵叶；

【导师】 陈代文； 毛湘冰；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 本试验旨在比较藏猪和DLY猪抗病毒相关模式识别受体基因表达差异,探明VD的抗病毒效果及其对基因表达的影响,以揭示不同品种猪抗病力差异和VD抗病的分子机制。首先应用生物信息学和荧光定量方法,在克隆了藏猪维甲酸诱导基因Ⅰ(RIG-Ⅰ)、β干扰素启动子刺激分子1(IPS-1)、Toll样受体3(TLR3)基因cDNA序列基础上,研究了RIG-Ⅰ、IPS-1和TLR3基因在藏猪和DLY猪不同组织的表达差异；然后建立了PRRS弱毒苗攻毒模型,研究藏猪和DLY猪抗病毒先天性免疫应答的差异；最后通过体外和体内试验探索了VD的抗病毒作用及其分子机制。本研究包括以下四个试验：试验一DLY猪和藏猪RIG-Ⅰ、IPS-1和TLR3基因表达差异的研究选取同一生长阶段、体重相近的纯种藏猪和DLY杂交猪各6头(公母各半),屠宰后取其心、肝、脾、肺、肾、十二指肠、空肠、回肠、支气管淋巴结、肠系膜淋巴结、腹股沟淋巴结、肌肉和脂肪等组织,提取总RNA,克隆藏猪RIG-Ⅰ、IPS-1和TLR3基因cDNA序列,用Real-time PCR方法检测RIG-Ⅰ、IPS-1和TLR3mRNA表达水平。结果如下：(1)藏猪RIG-Ⅰ基因cDNA序列长2832bp,包含从起始密码子1位到终止密码子2832位的开放阅读框,编码943个氨基酸残基,核苷酸序列与已报道猪的相似性为99.58%,氨基酸序列与人和鼠的相似性分别为77.67%和71.85%,不含跨膜区域。(2)藏猪IPS-1基因cDNA序列长1575bp,包含从起始密码子1位到终止密码子1575位的开放阅读框,编码524个氨基酸残基,核苷酸序列与已报道猪的相似性为99.37%,氨基酸序列与人和鼠相似性分别为55.04%和45.03%,含一个跨膜区域。(3)藏猪TLR3基因cDNA序列长2718bp,包含从起始密码子1位到终止密码子2718位的开放阅读框,编码901个氨基酸残基,核苷酸序列与已报道猪的相似性为99.56%,氨基酸序列与人和鼠相似性分别为83.43%和77.35%,含一个跨膜区域。(4)所有组织均能检测到RIG-Ⅰ、IPS-1和TLR3mRNA表达。藏猪大部分组织的RIG-!和IPS-1mRNA水平均高于DLY猪(P<0.01)。RIG-Ⅰ和TLR3基因在藏猪肝和十二指肠的表达量最高,RIG-Ⅰ和TLR3基因在DLY猪肝、空肠、肝和肾表达量最高。藏猪和DLY猪的IPS-1基因均在肝和肾表达量最高。试验二PRRS弱毒苗对DLY猪和藏猪RIG-Ⅰ、IPS-1和TLR3基因表达的影响选用同一生长阶段、体重相近的纯种藏猪(15.44±0.34kg)和DLY (15.21±0.30kg)猪各12头,试猪单笼饲养。试验采用2×2因子试验设计,分别为藏猪与DLY猪,PRRS弱毒苗攻毒与不攻毒。试验分为4处理,每个处理6个重复,每个重复1头猪。试验预试期7d,正式期13d。第14d早上空腹采血后屠宰。结果如下：(1)DLY猪的日增重高于藏猪(P<0.01), PRRS弱毒苗攻毒降低了DLY猪和藏猪的日增重和日采食量(P<0.01),提高了饲料增重比(P<0.01),对DLY猪生产性能的影响强于藏猪(P<0.01)。(2) PRRS弱毒苗攻毒提高两种猪血清中免疫球蛋白IgG和IgM的浓度(P<0.01),但与品种之间无交互效应(P>0.05)。(3) PRRS弱毒苗攻毒提高了细胞因子IL-1β(P<0.01)、TNF-α (P<0.05)和IFN-β(P<0.05)的浓度,对DLY猪血清中IL-1p和IFN-β的影响强于藏猪(P<0.05)。(4) PRRS弱毒苗攻毒能提高RIG-Ⅰ在脾(P<0.01)、肝(P<0.05)、肺(P<0.01)、支气管淋巴结(P<0.05)、肠系膜淋巴结(P<0.01)、腹股沟淋巴结(P<0.01)、空肠(P<0.01)和回肠(P<0.01)的表达,提高IPS-1在脾(P<0.01)、肝(P<0.01)、肺(P<0.01)、肠系膜淋巴结(P<0.01)、空肠(P<0.01)和回肠(P<0.05)的表达,提高TLR3在脾(P<0.01)、肝(P<0.01)、肺(P<0.05)、肠系膜淋巴结(P<0.01)、腹股沟淋巴结(P<0.01)、空肠(P<0.01)和回肠(P<0.01)的表达。(5) PRRS弱毒苗攻毒对DLY猪肺(P<0.01)、肠系膜淋巴结(P<0.05)和回肠(P<0.01)组织RIG-Ⅰ mRNA表达的影响强于藏猪；对DLY猪肝(P<0.01) IPS-1mRNA表达的影响强于藏猪；对DLY猪腹股沟淋巴结(P<0.01) TLR3mRNA表达的影响强于藏猪。试验三VD对感染RV IPEC-J2细胞系RIG-Ⅰ、IPS-1和TLR3基因表达与IFNβ蛋白表达的影响为了考察VD的抗病毒作用及其信号机制。本试验选用猪IPEC-J2细胞系为研究模型。病毒为猪轮状病毒OSU株。试验采用2x2x2因子试验设计,分别为感染RV与不感染,加入生理浓度(10-7M)25D3与不加25D3,加入CYP27B1酶阻断剂依曲康唑(ITRA,10-7M)与不加ITRA,,每个处理6个重复。24h后,提取总RNA,考察RIG-Ⅰ及其下游信号分子、TLR3mRNA表达,ELISA考察IFN-α蛋白含量。结果如下：(1)研究建立了RV感染IPEC-J2细胞模型。结果表明：RV感染IPEC-J2细胞24h后细胞体积缩小,连接消失,与周围的细胞脱离呈网状结构,当感染72h后细胞圆缩、坏死、脱落。通过MTS法检测细胞的增殖情况,RV感染极显著降低了细胞的增殖。RT-PCR检测感染RV的细胞均检测到RV RNA的存在。(2)RV感染提高了IPEC-J2细胞CYP27B1mRNA表达和上清液中1,25D3水平(P<0.01)。(3)RV感染提高RIG-Ⅰ、IPS-1、ISG15和IFN-β mRNA的表达(P<0.01),对TLR3mRNA的表达无显著影响(P>0.05)。VD提高RV感染IPEC-J2细胞RIG-Ⅰ、 IPS-1、ISG15和IFN-βmRNA的表达(P<0.01)。RV感染和VD互作对RIG-Ⅰ、IPS-1、 ISG15和IFN-p mRNA表达有显著影响,VD提高RIG-Ⅰ、IPG15和IFN-β mRNA表达的程度为感染组高于未感染组(P<0.01)。RV感染提高了IFN-β蛋白水平(P<0.01),VD提高了IFN-β蛋白水平(P<0.01),RV和VD互作对IFN-β蛋白有显著影响,VD提高IFN-β蛋白的程度为感染组高于未感染组(P<0.01)。试验四VD对感染RV藏猪和DLY猪肠道TLR3和RIG-Ⅰ及下游信号分子基因表达的影响为了考察动物试验中VD对RIG-Ⅰ及下游信号分子和TLR3基因表达的影响。选用同一生长阶段体重为13.73±0.95kg的纯种藏猪16头和DLY猪(26.46±2.07kg)24头,试猪单笼饲养。试验采用2×2x2因子试验设计,分别为藏猪与DLY猪,感染RV与不感染,基础日粮(VD为NRC水平)与基础日粮添加VD(VD为25倍NRC水平)。试验分为8个处理,藏猪每个处理4个重复,每个重复1头猪,DLY猪每个处理6个重复,每个重复1头猪。试验预试期7d,正式期6d,第7d早上空腹采血后屠宰。结果如下：(1)RV攻毒降低了猪平均日增重和日采食量(P<0.01),提高了料重比(P<0.01)；日粮添加VD提高了平均日增重和日采食量(P<0.01),对料重比无显著影响；RV攻毒对DLY猪生产性能的影响强于藏猪(P<0.01)。(2)RV攻毒降低了绒毛高度(P<0.01),对隐窝深度和绒隐比无显著影响；日粮添加VD有提高绒毛高度的趋势,但差异不显著,对隐窝深度和绒隐比无显著影响。品种和RV攻毒的互作对绒毛高度有显著影响,RV攻毒降低绒毛高度的程度为DLY猪显著高于藏猪。(3)RV攻毒提高了血清中IFN-β、IL-6和IL-2水平(P<0.01)；添加VD提高了血清IFN-β水平(P<0.01),降低了血清中1L,-6和IL-2水平(P<0.01)；品种与RV攻毒互作对IFN-β、IL-6和IL-2有显著影响,RV攻毒提高IFN-β、IL-6和IL-2的程度均为DLY猪高于藏猪(P<0.01)。(4)RV攻毒提高了十二指肠、空肠和回肠CYP27B1mRNA表达(P<0.01)；日粮添加VD提高了十二指肠、空肠和回肠CYP27B1mRNA表达(P<0.01),提高十二指肠CYP27B1mRNA表达的程度为DLY猪高于藏猪(P<0.01)；RV攻毒降低了血清中1,25D3水平(P<0.01)；添加VD提高了血清中1,25D3水平(P<0.01)。(5)RV攻毒提高了十二指肠、空肠和回肠RIG-Ⅰ、IPS-1、ISG15和IFN-β mRNA表达(P<0.01),对十二指肠、回肠TLR3mRNA表达无显著影响；日粮添加VD提高了十二指肠、空肠和回肠RIG-Ⅰ、IPS-1、ISG15和IFN-β mRNA表达(P<0.05),对十二指肠、空肠和回肠TLR3mRNA表达均无显著影响。综上所述,抗病力不同的藏猪和DLY猪体内RIG-Ⅰ、IPS-1和TLR3的表达水平存在明显的品种和组织差异；藏猪具有较强的疾病抵抗力可能与这些基因表达量高有关；VD具有一定的抗病毒功能,其机制与调节这些基因表达有关,且RIG-Ⅰ信号途径是其实现抗病毒作用的信号转导通路之一。更多还原

【Abstract】 The study was designed to investigate differences of messenger RNA expression for antiviral pattern recognition receptors between Tibetan and DLY pigs, the antiviral activity of vitamin D and the effect of vitamin D on gene expression in order to reveal the differences in disease resistance of different breeds of pigs, and vitamin D’s antiviral activity and mechanism. The present study firstly cloned and characterized Tibetan porcine retinoic acid-inducible gene Ⅰ (RIG-Ⅰ), interferon-beta promoter stimulator1(IPS-1) and Toll-like receptor3(TLR3) via the method of molecular cloning, bioinformatics and fluorescence quantitative PCR, and then investigate the tissue distribution and differences of messenger RNA expression for RIG-Ⅰ, IPS-1and TLR3between Tibetan and DLY pigs. Afterwardswe explored the difference of the innate antiviral immune response to PRRS virus between Tibetan and DLY pigs with PRRS vaccine challenge model. Finally, we explored the antiviral activity of vitamin D and molecular mechanism in vitro and in vivo. This study includes the following four experiments.Experiment1:Expression differences of RIG-Ⅰ, IPS-1and TLR3between DLY and Tibetan pigs.Six Tibetan pigs and six DLY pigs with half male and half female were slaughtered at the same growth stage and similar weight. The tissues, including heart, liver, spleen, lung, kidney, duodenum, jejunum, ileum, bronchial lymph nodes, mesenteric lymph node, inguinal lymph nodes, muscle and adipose tissue, were collected. Total RNA was extracted for cloning of Tibetan pig RIG-Ⅰ, IPS-1and TLR3gene cDNA sequence, and was used to detect RIG-I, IPS-1and TLR3gene expression by real-time quantitative PCR. The results are as follows:(1) The Tibetan RIG-I cDNA was successfully cloned. This sequence contains a2832bp opening reading frame, and encodes943amino acid residues. The nucleotide sequence shares99.58%homology with the known RIG-I sequences of the common pig. The AA sequence has77.67%and71.85%homology with human and mus musculus respectively. Hydrophobicity analysis of the AA sequence suggests no transmembrane region.(2) The Tibetan IPS-1cDNA contains a1575bp opening reading frame, and encodes524amino acid residues. The nucleotide sequence shares99.37%homology with the known IPS-1sequences of the common pig. The AA sequence has55.04%and45.03%homology with human and mus musculus respectively. Hydrophobicity analysis of the AA sequence suggests the presence of1putative transmembrane domain.(3) The Tibetan TLR3cDNA contains a2718bp opening reading frame, and encodes901amino acid residues. The nucleotide sequence shares99.56%homology with the known TLR3sequences of the common pig. The AA sequence has83.43%and77.35%homology with human and mus musculus, respectively. Hydrophobicity analysis of the AA sequence suggests the presence of1putative transmembrane domain.(4) RIG-I, IPS-1and TLR3are ubiquitously expressed in all tissues. The RIG-Ⅰ and IPS-1expression of most tissues of Tibetan pigs were higher than those of DLY pigs (P<0.01). The RIG-Ⅰ and TLR3expression of the liver and duodenum were highest for Tibetan pig. RIG-Ⅰ expression was highest in liver and jejunum of DLY pig. TLR3expression was highest in liver and kidney of DLY pig. The expression of IPS-1in the liver and kidney was highest for Tibetan and DLY pig.Experiment2:RIG-Ⅰ, IPS-1and TLR3gene expression of DLY pigs and Tibetan pigs after challenge with PRRS vaccine.Twelve Tibetan pigs (15.44±0.34kg) and twelve DLY pigs (15.21±0.3kg) were randomly allotted to4treatments with6replicates per treatment, and1pig per replicate. The experiment was designed by2×2factorial arrangement based on the factors of pig breed (Tibetan pigs and DLY pigs) and challenge status (PRRS vaccine challenge and unchallenged). The trial period included7days of preliminary experiment and13days of formal experiment. The results are as follows:(1) The ADG of DLY pigs was significantly higher than that of Tibetan pigs (P<0.01). After challenged with PRRS vaccine, ADG and ADFI were decreased, feed conversion rate was increased in Tibetan pigs and DLY pigs (P<0.01). The effect of PRRS vaccine on growth performance of DLY pigs was greater than that of Tibetan pigs (P<0.01).(2) The IgG and IgM concentrations of serum increased after challenge with PRRS vaccine in both breeds of pigs (P<0.01). But there is no interaction effect between breed and PRRS vaccine.(3) Serum IL-1β (P<0.01), TNF-α (P<0.05), and IFN-β (P<0.01) concentrations increased after challenge with PRRS vaccine. The effect of PRRS vaccine on IL-1β and IFN-β concentrations of DLY pigs were greater than that of Tibetan pigs (P<0.01).(4) The challenge with PRRS vaccine could increase the RIG-Ⅰ expression in liver (P<0.01), spleen (P<0.05), lung (P<0.01), bronchial lymph nodes (P<0.05), mesenteric lymph node (P<0.01), inguinal lymph nodes (P<0.01), jejunum (P<0.01) and ileum (P<0.01), stimulate the IPS-1expression in liver (P<0.01), spleen (P<0.01), lung (P<0.01), mesenteric lymph node (P<0.01), jejunum (P<0.01) and ileum (P<0.05), and enhance the TLR3expression in liver (P<0.01), spleen (P<0.01), lung (P<0.05), mesenteric lymph node (P<0.01), inguinal lymph nodes (P<0.01), jejunum (P<0.01) and ileum (P<0.01).(5) The effect of PRRS vaccine on lung (P<0.01), mesenteric lymph node (P<0.05) and and ileum (P<0.01) RIG-Ⅰ mRNA expression, liver IPS-1mRNA expression (P<0.01), and inguinal lymph nodes (P<0.01). TLR3mRNA expression of DLY pigs was greater than that of Tibetan pigs (P<0.01).Experiment3:The effect of vitamin D on RIG-I, IPS-1, and TLR3gene expression and IFN beta protein expression in RV infected IPEC-J2cellThe study evaluated whether vitamin D could decrease rotavirus replication potentially through RIG-Ⅰ signaling pathway in IPEC-J2cells. Porcine rotavirus OSU strain was used in this study. The experiment was designed by a2×2×2factorial arrangement based on the factors of infect status (RV infected and uninfected),25D3concentration (0and10-7M25D3), and ITRA concentration (0and10-7M ITRA). At24hours after treatment, total RNA was extracted. The expression of RIG-Ⅰ, IPS-1, and TLR3mRNA were determined by real-time PCR, and the protein levels of IFN-(3were measured by ELISA. The results are as follows:(1) The model of RV infected IPEC-J2cell was established. At24h after infected with RV, IPEC-J2cells were shrinked, and the connection among cells disappeared. At72h after infected with RV, IPEC-J2cells showed necrosis and shedding. The RV infection significantly decreased IPEC-J2cell proliferation. RV RNA were detected in RV infected IPEC-J2cells.(2) The CYP27B1mRNA in RV-infected cells was higher than that in control cells (P<0.01). The1,25D3level of the supernatant in RV-infected cells was higher than that in uninfected cells (P<0.01).(3) RV infection alone resulted in the increase in RIG-Ⅰ, IPS-1, ISG15and IFN-β expression (P<0.01), but did not affect TLR3expression (P>0.05). RIG-I, IPS-1, ISG15, IFN-P mRNA expression and IFN-p protein levels in IPEC-J2cells infected with RV and treated with25D3(10-7M) showed a significant increase. The effect of vitamin D on RIG-Ⅰ, IPS-1, ISG15, IFN-β mRNA expression and IFN-P protein levels of RV infected IPEC-J2cell was greater than that of uninfected cell (P<0.01).Experiment4:The effect of vitamin D on RIG-Ⅰ and its downstream signaling molecules, TLR3mRNA expression in RV infected Tibetan and DLY pigsThe study evaluated whether vitamin D could decrease rotavirus replication potentially through RIG-Ⅰ signaling pathway in vivo. The experiment was designed by2×2×2factorial arrangement based on the factors of pig breed (Tibetan pigs and DLY pigs), challenge status (RV challenged and unchallenged), VD concentration (200IU and5000IU). Sixteen Tibetan pigs (13.73±0.95kg) and twenty four DLY pigs (26.46±2.07kg) were randomly allotted to8treatments with6replicates per treatment for DLY pigs, and4replicate per treatment for Tibetan pigs. The trial period included7days of preliminary experiment and6days of formal experiment. The results are as follows: (1) ADG and ADFI were decreased, and F/G was elevated by RV challenge (P<0.01). Dietary supplementation with vitamin D elevated ADG and ADFI (P<0.01). The effect of RV challenge on growth performance of DLY pigs was greater than that of Tibetan pigs (P<0.01).(2) Villus height was decreased by RV challenge (P<0.01). There was not effect on crypt depth and the villus height:crypt depth ratio by RV challenge (P>0.05). Dietary supplementation with vitamin D elevated villus height, whereas there was no significantly difference. The effect of RV challenge on villus height of DLY pigs was greater than that of Tibetan pigs (P<0.01).(3) Serum IFN-(3, IL-6and IL-2concentrations decreased by RV challenge (P<0.01). The effect of RV challenge on serum IFN-β,IL-6and IL-2concentrations of DLY pigs was greater than that of Tibetan pigs (P<0.01). Dietary supplementation with vitamin D elevated IFN-β level, and decreased IL-6and IL-2concentrations in serum (P<0.01).(4) The expression of CYP27B1mRNA in duodenum, jejunum and ileum increased by RV challenge and vitamin D treatment (P<0.01). The effect of dietary vitamin D levels on CYP27B1mRNA expression of DLY pigs was greater than that of Tibetan pigs. Serum1,25D3concentrations decreased by RV challenge (P<0.01), but elevated by dietary supplementation with vitamin D (P<0.01).(5) The expression of RIG-Ⅰ, IPS-1, ISG15and IFN-P mRNA in duodenum, jejunum and ileum increased by RV challenge and dietary supplementation with vitamin D (P<0.01). But there was no difference in TLR3gene expression by RV challenge and dietary supplementation with vitamin D (P>0.05).In summary, the expression distribution of RIG-Ⅰ, IPS-1and TLR3mRNA in Tibetan and DLY pigs was different. The higher expression of RIG-Ⅰ, IPS-1might be the reason that Tibetan pigs has higher immunity and disease resistance. Vitamin D had certain inhibiting ability for rotavirus. The anti-rotavirus effect of vitamin D was mediated at least in part by RIG-Ⅰ signaling pathways in IPEC-J2cells.更多还原