谷跗线螨的形态学观察、抗原定位及精氨酸激酶基因的克隆与序列分析

发布时间：2018-01-06 22:20

本文关键词：谷跗线螨的形态学观察、抗原定位及精氨酸激酶基因的克隆与序列分析　出处：《蚌埠医学院》2012年硕士论文　论文类型：学位论文

【摘要】：由变应原引起的变态反应性疾病(如哮喘、变应性鼻炎等)是当前世界性的重大卫生学问题，总发病率高达10％～30％，并有逐年增多的趋势。谷跗线螨作为一种常见的仓储螨类，以往人们对其关注较少，其形态特征、组织结构特点和生物学特性尚不十分明了。为进一步明了谷跗线螨的基本生物学尤其是其显微形态特点，探讨其与粉尘螨抗原的交叉反应性，并尝试探讨其致敏原的相关分子，本研究进行了以下三方面的工作：（1）谷跗线螨的扫描电镜观察及其内部主要器官的观察：从空调滤网灰尘中采集谷跗线螨，在体视显微镜下清洗处理活螨后，用SEM观察其外部形态特征。经形态鉴定确认为该螨种后，以Bouin液固定，制作谷跗线螨石蜡切片，HE染色后光镜观察其内部形态结构。结果清晰地观察到谷跗线螨的体表形态以及主要的内部器官的构造，明确了谷跗线螨颚体、背板、基节板、表皮内突、背足体、刚毛等外部形态以及其消化道等的内部结构特点。（2）谷跗线螨与粉尘螨交叉反应性变应原的定位研究：制作谷跗线螨石蜡切片，先用对粉尘螨过敏患者的阳性血清作为一抗孵育，再用生物素标记的羊抗人IgE抗体作为二抗，免疫组织化学染色后在光学显微镜下分析其交叉反应性抗原存在位置。研究发现谷跗线螨口咽部、中肠、肠内容物、肠壁及生殖腺等均有阳性反应。研究表明谷跗线螨体内存在可诱发人体IgE抗体的变应原，主要分布于螨消化道、腺体及体壁等部位。（3）谷跗线螨精氨酸激酶（arginine kinase,AK）基因的克隆及序列分析：从空调滤网灰尘中采集谷跗线螨（约3000只）提取总RNA，，经逆转录RT－PCR扩增谷跗线螨AK基因后测序。克隆获得的谷跗线螨AK基因由724个碱基组成，其编码的蛋白相对分子质量约为26019，等电点为10.10。成功克隆了谷跗线螨AK基因，为进行谷跗线螨AK蛋白的重组表达以及过敏原性分析奠定基础。上述研究积累了有关谷跗线螨显微形态、与已知过敏原的交叉反应性和过敏原相关分子研究的新资料，为今后谷跗线螨过敏性疾病的特异性诊断、治疗和预防奠定工作基础。
[Abstract]:Allergic diseases ( such as asthma , allergic rhinitis , etc . ) caused by allergens are the major health problems in the world . The total incidence is as high as 10 % -30 % , and there is a tendency to increase year by year . In order to further clarify the basic biology , especially its micro - morphological characteristics , the cross - reactivity with dust mite antigen was discussed , and its related molecules were tried to explore its allergen . The study carried out the following three aspects : ( 1 ) Scanning electron microscope ( SEM ) and observation of main organs were observed . After the mites were collected from the dust of the air - conditioning filter screen , the morphological characteristics of the mites were observed by SEM . ( 2 ) The localization of cross - reactive allergens of tarsomite and dust mites was studied in this paper . The results showed that there were positive responses to the cross - reactive antigens in the mite mouth pharynx , midgut , intestinal contents , intestinal wall and reproductive gland . ( 3 ) Cloning and sequence analysis of the arginine kinase ( AK ) gene of tarsomite : A total RNA was extracted from the dust of the air - conditioning filter screen . The total RNA was amplified by reverse transcription RT - PCR . The AK gene was amplified by RT - PCR . The relative molecular weight of the protein was about 26019 and the isoelectric point was 10.10 . The AK gene was successfully cloned , which laid the foundation for the recombinant expression of AK protein and the analysis of allergen . The above studies have accumulated new information on the micromorphology of tarsonemus , cross - reactivity with known allergens and molecular studies of allergen - related molecules , laying the foundation for the specific diagnosis , treatment and prevention of allergic diseases in the future .

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】