【作者】 王开胜；

【导师】 贾斌；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2017， 博士

【摘要】 目的:新疆外引绵羊育种实践表明,湖羊对绵羊支原体肺炎较为易感,其感染率和死亡率一直保持在较高水平,而同为引进品种的杜泊羊对该病的抗病力较强,同时杜泊羊与湖羊的杂交一代绵羊也很好地表现出其父本杜泊羊较强抗病力的特性。研究证明,绵羊MHC多态性与多种疾病有一定相关性,其中,OLA-DRB基因具有丰富的多态性,MHCⅡ类分子的DR亚区则是MHC抗性/易感性研究主要区域之一。绵羊的MHC-DRB1基因外显子2的基因杂合度较大,从而表现为其遗传变异程度较高。因此,本研究在前期基因分型研究的基础上,对湖羊、杜泊羊及杜湖F₁代绵羊的MHC基因多态性与其对支原体肺炎的抗性/易感性的关联性进行研究。此研究在遗传学方面对新疆引进绵羊品种的抗病育种研究具有一定的意义,可提供理论和实践参考依据。方法:利用聚合酶链式反应-限制性片段长度多态性（PCR-RLFP）方法对新疆引进品种杜泊羊、湖羊及杜湖F₁代绵羊群体的MHC-DRB1基因外显子2进行PCR-RFLP多态性分析,以确定其与绵羊肺炎支原体抗性、易感性相关的候选基因型。本研究选择了绵羊支原体肺炎发病率较低且兼具父母本遗传特性、遗传背景相对一致的杜湖F₁代羊为研究对该病易感与抗性基因的最佳群体,因此,挑选出候选易感、抗性基因型绵羊个体进行分组,人工感染绵羊肺炎支原体前后测量实验羊体温,观察其临床症状,进行肺脏组织病理变化和组织病理学变化评分;采用ELISA诊断试剂盒检测感染前后各组绵羊细胞免疫因子（TNF-α、IFN-γ、IL-1β、IL-2）和体液免疫因子（IL-4、IL-6、IL-10）蛋白表达变化;采用qRT-PCR检测ELISA细胞免疫因子和体液免疫因子mRNA的表达变化,从而验证绵羊支原体肺炎抗性和易感性候选基因型与该病的关联性。结果:（1）绵羊支原体肺炎易感群体湖羊、抗病力较强的杜泊羊群体及杜湖F₁代绵羊群体的MHC-DRB1基因外显子2均可被SacⅠ、HaeⅢ、SacⅡ、MvaI和BsaHⅠ五种限制性核酸内切酶检测出多态性。三群体绵羊的SacⅠ、SacⅡ、MvaI和Bsa HⅠ酶切位点均由双等位基因控制,分别表现出3种基因型。HaeⅢ限制性核酸内切酶在三群体中表现出较大差异,对绵羊支原体肺炎较易感的湖羊群体有7个等位基因,表现出15种基因型;抗病力较强的杜泊羊群体和杜湖F₁代绵羊群体均受到6个等位基因控制,分别表现出14、11种基因型。绵羊支原体肺炎抵抗力较弱的湖羊群体与抵抗力较强的杜泊羊和杜湖F₁代绵羊群体MHC-DRB1外显子2在Mva I与HaeIII酶切多态中差异极显著（P<0.01）,Mva I bb、HaeIII ee基因型均出现在绵羊支原体肺炎抵抗力较弱的湖羊群体,而抵抗力较强杜泊羊及杜湖F₁代绵羊群体则以MvaI cc、Hae III dd基因型为优势基因型。提示Mva I cc、HaeIII dd基因型可能与绵羊支原体肺炎具有较强的相关性。（2）在对杜泊羊、湖羊及杜湖F₁代绵羊群体MHC-DRBl多态性及其绵羊支原体肺炎抗性/易感相关分析的基础上,根据分析得出与该病抗性关联的基因单倍型,从杜湖F1代绵羊群体中挑选出可能与绵羊支原体肺炎抗性相关的HaeIII dd和Mva I cc基因型健康绵羊各5只,分别为A组和B组,易感关联HaeIII ee基因型健康绵羊5只,为C组。人工感染绵羊支原体肺炎前后的结果显示:3组绵羊体温变化显示出起伏的变化,A组（HaeIII dd基因型）和B组（Mva I cc基因型）绵羊的体温变化趋势较为一致,C组（HaeIII ee基因型）在1 d内的体温差极显著高于A组和B组（P<0.01）。肺组织病理变化和组织病理学评分结果显示:C组绵羊的感染评分明显高于A组和B组（P<0.05或P<0.01）,而A组和B组之间的差异不显著（P>0.05）。经对3组绵羊的体温变化、临床症状和组织病理变化评分和组织病理学评分进行分析,A组和B组绵羊对绵羊支原体肺炎的抗性强于C组,即HaeIII dd和Mava I cc基因型绵羊的抗性明显强于Hae III ee基因型绵羊。（3）为了探明在感染绵羊肺炎支原体前后3个基因型组杜湖F₁代绵羊体内免疫因子的应答情况,对其血清中的免疫因子水平进行检测。结果表明:（1）在感染后,TNF-α、IFN-γ、IL-1β、IL-2四个免疫因子的水平整体上均呈现出先升高后下降的趋势。A组和B组绵羊血清中细胞因子的出现峰值时间早于C组,且持续时间较长。（2）体液免疫因子的检测结果显示,在感染后,IL-4、IL-6、IL-10三个免疫因子的水平整体上均呈现出先升高后下降的趋势。A组和B组的三个免疫因子的出现峰值时间早于C组,且持续时间较长。在人工感染绵羊肺炎支原体后,C组绵羊体内Th1型细胞因子（TNF-α、IFN-γ、IL-1β、IL-2）的表达量较A组和B组显著降低（P<0.05）,但其Th2型细胞因子（IL-4、IL-6、IL-10）的表达量较A组和B组显著升高（P<0.05）。这与C组绵羊感染绵羊肺炎支原体后病情加重、病变显著的结果一致。表明A组（HaeIII dd基因型）与B组（Mva I cc基因型）表现为抗性,可以激活细胞免疫应答反应,降低炎症反应,从而保护羊抵抗绵羊肺炎支原体的感染。（4）人工感染绵羊肺炎支原体前后,3组绵羊的免疫因子mRNA表达水平结果显示,TNFαmRNA表达水平均呈现先上升后下降的趋势,第7天均达到峰值;IL-2 mRNA表达水平呈现先上升后下降的趋势,其中以感染后第14天水平最高;IFN-γmRNA表达水平呈现为先下降后升高,但低于感染前的水平;IL-4 mRNA表达显著升高,而IFN-γmRNA表达显著下降,IL-4/IFN-γ比值增高。C组的IL-4/IFN-γ比值的增高明显高于A组和B组（P<0.05）,说明C组的病情较A组和B组更加严重;感染后第7天开始,C组IL-6水平始呈上升趋势,且显著高于A组和B组（P<0.05）;C组IL-10 mRNA表达水平一直低于A组和B组。各免疫因子的mRNA表达水平的变化说明了A组和B组表现出的抗性强于C组,说明A组（Hae III dd基因型）与B组（Mva I cc基因型）绵羊机体的免疫力及抗病力强于C组（Hae III ee基因型）。结论:（1）本研究分析了3个群体绵羊MHC-DBR1基因第二外显子5个核酸内切的PCR-RLFP分析数据,发现绵羊支原体肺炎高发的湖羊群体与低发杜泊羊群体在HaeIII与Mva I内切酶酶切多态上存在较大差异,提示HaeIII ee、MvaI bb基因型绵羊可能易感肺炎支原体肺炎,而Hae III dd、Mva I cc基因型绵羊可能对该病具有抗性。（2）对A组（Hae III dd基因型）、B组（Mva I cc基因型）和C组（HaeIII ee基因型）绵羊进行绵羊肺炎支原体人工感染,结果表明,A组和B组绵羊在体温变化、临床症状和组织病理变化评分和组织病理学评分中均优于C组,提示A组和B组绵羊对新疆本地气候的适应性和抗病力强于C组。（3）绵羊肺炎支原体攻毒后,A组和B组绵羊的各免疫因子的蛋白与mRNA表达水平均与C组存在差异,C组绵羊体内Th1型细胞因子（TNF-α、IFN-γ、IL-1β、IL-2）的表达量较A组和B组显著降低（P<0.05）,但其Th2型细胞因子（IL-4、IL-6、IL-10）的表达量较A组和B组显著升高（P<0.05）。人工感染绵羊肺炎支原体试验验证了抗性基因型（HaeIII dd、Mava I cc）组绵羊对绵羊支原体肺炎的免疫力及抗病力强于易感基因型（HaeIII ee）组绵羊。更多还原

【Abstract】 Object:The breeding practice of sheep introduced from outside showed that Hu sheep is more susceptible to Mycoplasmal pneumonia of sheep and the rates of infection and mortality remained at a high level,but the same introduced variety Dorper sheep showed stronger resistance to the disease.Hybrid F₁ generation of Dorper and Hu sheep maintained the paternal Dorper sheep characteristics of strong resistance to the disease.Many studies proved that MHC polymorphism in sheep is associated with a variety of diseases.Among them,OLA-DRB gene is rich in polymorphism,and the DR subunit of MHC class II molecules is one of the main research areas for the study of MHC resistance/susceptibility.The gene heterozygosity of the MHC-DRB1 gene exon 2 in sheep is high,and shows high degree of genetic variation.In the present study,we studied the sheep MHC gene polymorphism of Hu sheep,Dorper sheep and their Hybrid F₁ based on early genotyping.We performed association analysis of MHC gene polymorphism with the resistance/susceptibility to Mycoplasma pneumonia of sheep.This study has acertain significance in genetics for the introduction of sheep breeding for disease resistance in Xinjiang,and can provide theoretical and practical references.Method:Using PCR-RLFP method,the polymorphism for the second exons of the MHC-DRB1 gene of Dorper sheep,Hu sheep and the hybrid F₁ generation of Dorper and Hu sheep were analyzed,and the candidate genotypes related to resistance or susceptibility to Mycoplasma pneumoniae of sheep were determined.In this study,the F₁ generation sheep with a low incidence of the disease and a relatively consistent genetic background were selected as the best group to study the susceptible and resistant genes of the disease.Therefore,the candidate susceptibility,resistant genotypes of sheep were grouped;the body temperatures of the experimental sheep were measuredand their clinical symptoms were observed;their histopathological changes were scored before and after artificially infected with Mycoplasma pneumonia of sheep.The protein expression changes of the cellular immune factors（TNF-α,IFN-γ,IL-1β,IL-2）and the expression changes of humoral immune factors（IL-4,IL-6,IL-10）were detected using ELISA diagnostic kit before and after infection;the mRNA expression changes of the cellular immune factors and the humoral immune factors were detected by qRT-PCR method.These will verify the association of the resistance/susceptibility candidate genotypes with disease.Result:（1）The second exon polymorphisms of the MHC-DRB1 genes of the contagious Hu sheep,the resistant Dorper sheep and their F₁ sheep groups could be dectected by five restriction endonucleases of Sac I,Sac II,Hae III,MvaI and BsaHI.All the four-restriction enzyme cutting sites of Sac I,SacII,Mva I and BsaHI,enzymes of the three-group sheep were controlled by two alleles,and showed three genotypes.Hae III restriction endonuclease in the three groups showed bigger differences among the three groups,the susceptible Hu groups had 7 alleles and 15 genotypes;the resistant Dorper sheep group and their F₁ sheep had six alleles,and they had 14,11 genotypes,respectively.The groups of Hu sheep,Dorper sheep and the F₁sheep had significant difference（P<0.01）in Mva I and HaeIII polymorphic enzymes of the second exons,and Mva I bb and Hae III ee genotypes were found in Hu sheep group.However,Mva I cc,HaeIII dd genotypes were the dominant genotypes in Dorper sheep and the F₁ sheep.As a result,the genotypes of Mva I cc and HaeIII dd may be closely linked with resistance to Ovine pleuropneumonia of sheep.（2）On the basis of the association analysis of MHC-DRBl polymorphism and resistance/susceptibility of Dorper sheep,Hu sheep and the F₁ sheep to Mycoplasma pneumonia of sheep according to the disease resistance gene haplotype association analysis,five healthy F₁ sheep with HaeIII dd and Mva I cc genetypes possibly associated with the disease resistance were selected,and they were grouped A（HaeIII dd genetype）and B group（Mva I cc genetype）;five healthy F₁ sheep assoctated with Hae III ee genotype possibly associated with the disease susceptibility were selected,and they were grouped C（HaeIII ee genetype）.The results showed that the body temperature changes of the three groups before and after infection of Mycoplasma pneumonia of sheep showed fluctuations,the temperature-change trend of the A and B groups was more consistent,and the temperature difference in the C group was significantly higher than that in the A and B groups（P<0.01）in one day.The results of the histopathology changes and histopathological sections of the lungs showed that the infection score of the C group was significantly higher than that of the A and B groups（P<0.01）,but the difference between the A group and the B group was not significant（P>0.05）.The changes of body temperature,the scores of clinical symptoms,histopathological changes and histopathological sections in the three groups were analyzed,and the results showed that the resistance of the A and B groups to the disease was stronger than that of the C group;namely,the resistance of HaeIII dd and Mava I cc genotype sheep was significantly stronger than that of Hae III ee genotype sheep.（3）To explore the responses to the immune factor concentrations,the serum of the three genotypes of the F₁ sheep was detected before and after the infection.The detection results showed that:（1）the concentration levels of the four cell immune factors（TNF-α,IFN-γ,IL-1β,IL-2）in the three group rose first and then declined.The concentration peak times of A and B groups were earlier than that of the C group,and so was their duration time.（2）the concentration levels of the three humoral immune factors（IL-4,IL-6,IL-10）in the three group all rose first and then declined.The concentration peak times of the A and B groups were earlier than that of the C group,and so was their duration time.After artificial infection,the expression of the Th1 type cytokine（TNF-α,IFN-γ,IL-1β,IL-2）in the C group was significantly lower than that in the A and B groups（P<0.05）,but the expression of the Th2type cytokine（IL-4,IL-6,IL-10）was significantly higher than that in the A and B groups（P<0.05）.This was consistent with the severity of the disease and significant lesionsin of the C group infected with Mycoplasma pneumoniae of sheep.This indicated that the A group（HaeIII dd genotype）and the B group（Mva I cc genotype）showed resistance to the disease,can activate the immune response,reduce inflammation,and thereby protect the sheep against infection of the disease.（4）The mRNA detection results of the immune factors showed that before and after infection,the TNFαexpression level of 3 groups showed a trend of rising first and then decreasing,and all peaked d on the seventh day;IL-2 expression level first increased and then decreased,and reached the highest level in the fourteenth day;the expression level of IFN-γdecreased first and then increased,but lower than that before infection;IL-4 expression level was significantly increased,while theexpression of IFN-γdecreased significantly（P<0.05）,and the ratio of IL-4/IFN-γincreased（P<0.05）.The IL-4/IFN-γratio of the C group increased significantly higher than that of the A and B groups（P<0.05）,and this indicated that the condition of C group was more serious than that of A group and B group;in seventh day,expression of IL-6 of the C group was increased,and it was significantly higher than that of the A and B groups（P<0.05）;Expression of IL-10 the C group was lower than the A and B groups.Expression change of the immune factors showed that the A and B groups had better resistance than the C group,which indicated that the sheep with HaeIII dd and MvaI cc genotypes were stronger than those with HaeIII ee genotype in immunity and disease resistance.Conclusion:（1）In this study,we analyzed the five endonucleases data of MHC-DBR1 gene exon 2 of sheep from three groups,found that the Dorper sheep group with high incidence to the disease and the Hu sheep group with low incidence had a big difference in Mva I polymorphism cut enzyme,and this suggested that the sheep with HaeIII ee and Mva I bb genotypes were susceptible to the disease,while the sheep with HaeIII dd and MvaI CC genotypes were resistant.（2）After artificial infection by the disease,the body temperature,the scores of clinical symptoms,histopathological changes and histopathological sections in the A and B groups were better than that of the C group,which indicated that the adaptability and resistance of the A and B groups to the Xinjiang climate were stronger than that of the C group.（3）After infection,expression changes of genes and proteins for each immune factor of the A and B groups were different from the C group;the Th1 type cytokine（TNF-α,IFN-γ,IL-1β,IL-2）expressions of the C group were significantly lower than those of the A and B groups（P<0.05）,but the Th2 type cytokine（IL-4,IL-6,IL-10）expressions of the C group were significantly lower than those of the A and B groups（P<0.05）.The artificial infection experiment confirmed that the resistant genotype（HaeIII dd,Mava I cc）groups were stronger than that of the susceptible genotype（HaeIII ee）group to the disease in the immunity and resistance.更多还原