【作者】 彭秀丽；

【导师】 毕丁仁； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 鸡毒霉形体(Mycoplasma gallisepticum,MG)是鸡慢性呼吸道病的病原菌。本病在世界各地的鸡群中普遍存在,给养禽业造成严重的经济损失。许多研究表明,MG通过表面的黏附素(Protein Mycoplasma Gallisepticum Adheain,pMGA)与鸡呼吸道粘膜表面的受体结合而侵染宿主。为了阐明pMGA的结构、功能及其在鸡毒霉形体感染中的分子致病机制,本研究鉴定了鸡毒霉形体黏附素特性、探讨了pMGA与宿主受体分子的相互作用及受体在鸡组织中的分布规律,为进一步研究鸡体内pMGA受体基因的类型、分子结构及作用方式奠定了基础,取得了如下结果。1.鸡毒霉形体黏附素特性鉴定通过生物学软件及相关在线分析工具,对pMGA进行基于氨基酸序列的生物信息学分析,包括蛋白质理化特性、功能域、磷酸化位点、基序及空间结构等。分析表明,pMGA1.2主要在细胞外发挥生物学作用,分子量64.9kD,理论等电点为5.58,半衰期体外培养的哺乳动物网状细胞为30h,酵母菌体内20h以上,大肠杆菌体内10h以上;a螺旋占26.59%,主要存在于210位氨基酸之前;β折叠占23.08%,存在于210位氨基酸之后,无规卷曲占50.33%,该蛋白为稳定的球状蛋白质;pMGA1.2分子有6类基序模式,53个基序位点和21个抗原决定簇,而且有4处具有明显的卷曲螺旋;pMGA1.2含有5个结构域,分别为蛋白家族Myco_haema结构域、2个FIVAR结构域、C末端有1个甲基化-DNA-蛋白半胱氨酸甲基转移酶结构域和1个半乳糖结合区;pMGA1.2属于霉形体血凝素超家族的一员,与霉形体R株黏附蛋白相似度高达99%。2.鸡毒霉形体黏附素截短蛋白及全蛋白表达产物免疫学特性鉴定对pMGA1.2和pMGAⅣ片段基因原核表达条件进行优化,获得了pMGA1.2及pMGAⅣ可溶性表达的最佳条件。pMGA1.2表达量在裂解液全菌体和上清液中分别达18.5%和17%,pMGAⅣ表达量在裂解液全菌体和上清液中分别达28%和18%。采用GST亲和层析柱纯化pMGA1.2及pMGAⅣ,纯度达95%左右。免疫印迹结果表明,pMGA1.2和pMGAⅣ融合蛋白能与兔源鸡毒霉形体阳性血清发生特异性免疫反应,出现明显的显色条带;空载体对照无显色条带;表明本实验所获得的pMGA1.2及pMGAⅣ融合蛋白均具有良好的免疫学活性。3.免疫荧光检测pMGA受体在鸡胚组织中的分布用蛋白酶thrombin切去融合蛋白pMGA1.2的GST标签,获得纯化的pMGA1.2,建立了检测鸡胚组织中pMGA受体的间接免疫荧光组织化学方法。将20日龄SPF鸡胚的气管、心、肺、肝、肾、十二指肠、法氏囊、腺胃、胸腺和脾脏等组织制成石蜡切片,采用纯化的pMGA1.2与各组织作用,用兔源的MG阳性血清和FITC标记的羊抗兔IgG来检测受体分布。结果表明:除脾脏外,其他组织中均出现明亮的特异性荧光,说明这些组织可能均存在能与pMGA1.2特异性结合的蛋白即受体蛋白。其中,pMGA的受体分布于气管和十二指肠的纤毛上皮细胞,肺脏的肺泡上皮细胞,法氏囊盲突的淋巴小结的皮质区,胸腺的皮质部,腺胃的粘膜层,肝脏、心脏及肾脏中pMGA受体的位置暂时无法确定。4.鸡胚组织膜蛋白与鸡毒霉形体黏附素特异结合的研究采用差速离心提取鸡胚组织膜蛋白,经处理后固定于硝酸纤维素膜上。以纯化的pMGA1.2及pMGAⅣ为“靶蛋白”,用兔源MG阳性血清与羊抗兔酶标IgG检测鸡胚各组织中是否存在能与pMGA反应的组织蛋白;SDS-PAGE分析组织膜蛋白。结果表明,除脾脏外,其他组织中均存在与pMGA1.2和pMGAⅣ特异结合的膜蛋白,其中分子量为36.5kD或(和)27.2kD的蛋白可能是pMGA1.2的受体,pMGAⅣ是与受体结合的肽段。更多还原

【Abstract】 Mycoplasma gallisepticum(MG) is the etiologic agents of chronic respiratory disease which commonly occurred in the world and resulted in severe lose to poultry industry. Researches had indicated that MG infested host by connecting its protein of Mycoplasma Gallisepticum Adhesin(pMGA) with surface receptors on the mucosa of respiratory tract. To eliminate pMGA structure,function and molecular pathogenic mechanism of MG,the characteristics of pMGA was identified,and the interaction of pMGA with receptor in host was discussed.Moreover,receptor distribution in chick tissues was detected.These results including below,will be helpful to research the genotype,molecular structure and acting mode of pMGA receptor.1.Characteristic Identification of Mycoplasma Gallisepticum Adhesin ProteinBased on amino acid sequence,bioinformatics analysis of pMGA physical and chemical characteristics,domain,phosphorylated sites,sequent and space structure were carried out with biological software and online analytical tool.pMGA1.2 bio-function mainly occurred out of cell.Its molecular weight was 64.9kD,isoelectric point in abstracto was 5.58,estimated half-life were 30 hours,20 hours and 10 hours respectively in mammal reticulocyte,yeast and E.coli.pGMA1.2 was stable globular protein because of that its secondary structure had 26.5%a-helix mainly in front of the 210 amino acids, 23.08%βfolds and 50.33%andom coil mainly behind the 210 amino acids.There were 6 motif modes,53 motif sites,21 antigen determinants and 4 obvious coiled coils in pMGA1.2,pGMA1.2 also included a typical domain of the mycoplasma hemaglutinin superfamily,two FIVAR domains(Uncharacterized Sugar-binding Domain),one domain of methylated DNA-protein cysteine methyltransferase of C-terminal,one galactose binding domain-like.pGMA was also a member of MG hemagglutinin superfamily and had 99%semblance with adhesin protein sequence in R strain mycoplasma.2.Immunological Characteristics of Expression Product of Brachytmema and Complete Proteins of Mycoplasma Gallisepticum AdhesinUnder the optimum conditions,the soluble expression quantities of pMGA1.2 were highly to 18.5%and 17.0%in complete thaline and supernatant respectively,and expression amount of pMGAⅣwere up to 28%and 18%respectively in complete thaline and supematant.95%purity of pMGA1.2 and pMGAⅣwere obtained with GST affinity column.Obviously colored straps emerged with immunoblotting while fusion proteins of pMGA1.2 and pMGAⅣreacted with specific positive sera of MG,demonstrated that fusion proteins of pMGA1.2 and pMGAⅣhad satisfactory Immunologic competence.3.Receptor Distribution of pMGA in Chick Embryo Tissues with ImmunofluorescenceIndirect immunofluorescent histochemistry test was set up with purified pMGA1.2 separated from pMGA1.2·GST,in which the GST was cut off with Thrombin.Paraffin sections of tissues were respectively reacted with pGMA1.2,MG sera from rabbits and secondary antibodies marked with FITC in proper order.Beside spleen,specific fluorescence could be observed in trachea,lung,liver,kidney,duodenum,bursa of Fabricius,glandular stomach,thymus and heart from twenty days old SPF chick embryo, which suggested that specific pMGA1.2 receptors could reside in all these nine tissues or organs.Moreover,pMGA receptors mainly located on lymphatic nodule cortical area of diverticule of bursa of Fabricius,thymic cortical part,mucosa of glandular stomach,and ciliated epithelial cells of trachea,bronchus and duodenum.However,the distributed site of pMGA receptor in liver,heart and kidney couldn’t be defined still.4.Specific Combination of pMGA with Membrane Proteins in Chick Embryo TissuesMembrane proteins was abstracted with differential centrifugation,and doted on nitrocellulose filter.As target proteins,purified pMGA1.2 and pMGAⅣwere connected with membrane proteins and antibodies of MG from rabbits,and MG antibodies were combinated with IgG marked with HRP.SDS-PAGE was also applied to identify receptor proteins.The results showed that the membrane proteins with molecular weights 36.5kD and/or 27.2kD,specifically binding with pMGA1.2 and pMGAⅣ,might be receptor proteins of pMGA1.2,but pMGAⅣwas a peptide connecting receptor proteins and pMGA1.2.更多还原