伪旋毛虫新生幼虫cDNA文库的免疫筛选及序列分析

发布时间：2018-04-16 23:37

本文选题：伪旋毛虫 + 新生幼虫cDNA文库　；参考：《吉林大学》2012年硕士论文

【摘要】：旋毛虫病(trichinosis/trichinellosis)是一种人兽共患性食源性寄生虫病，由旋毛线虫感染引起，分布范围极其广泛，集中发生在东南亚欠发达国家和有打猎习惯的少数发达国家。近年来有关该病的报道大幅度提升，原因可能是随着生活水平的提高，人们对肉类食品的需求量也有所增加，跨区域性旅游等行为也导致该病的分布范围扩散。在北美洲国家、欧洲国家和澳大利亚等地，牛肉、马肉和熊肉等其它肉类的消费产品也是导致人类旋毛虫感染的重要原因。在中国东北三省，广西，西藏及云南等地，由于当地饮食习惯而引发的发病报道也时有发生，对社会造成严重的恐慌，由此造成的社会影响极其严重。在旋毛线虫属中已报道十二个种中，伪旋毛虫是唯一一种既可以感染哺乳动物也可以感染鸟类的旋毛线虫，流动性较大，，难防难控，有着异于其它种的特殊的流行分布及感染特点。成虫时期在宿主肠道侵袭时间持续长达30d，所产的新生幼虫有长达4个月的肌肉侵袭期，并且感染整个肌肉细胞，给感染者带来长期持久的疼痛。其诊断方式，主要是直接肌肉压片镜检或肌肉消化后用光学显微镜观察，由于伪旋毛虫在肌肉感染时期不形成包囊或仅形成包囊较薄，所以传统的肌肉压片镜检方法很难检测出该病原；另外由于道德伦理等方面的限制，在人体做肌肉切片检查也难以实现。伪旋毛虫发育不同时期体表抗原和分泌抗原各不相同，均能诱导宿主产生不同程度的免疫力。做为宿主免疫系统应答是别的主要针对时期，在血液中游走的新生幼虫是其整个生活史中唯一不在宿主细胞内寄生于的发育阶段,该时期虫体对宿主的免疫应答反应有着重要调节作用。伪旋毛虫肌肉幼虫在宿主肌肉细胞内生存时间长达数年甚至更久，在宿主体内产生的抗体可以用以诊断。而如果该时期的抗体能同时识别新生幼虫时期和成虫时期的虫体抗原，那么将寻找到一种识别虫体整个生活周期的特异性基因。本实验所用的伪旋毛虫(ISS13)新生幼虫cDNA文库，经过滴度测定，确定其滴度为4.25×107pfu/mL，完全符合实验标准。在对表达文库进行免疫筛选之前，必须首先扩增非特异性蓝斑并测定扩增后的蓝斑滴度，在达到一定的数量级后（109pfu/mL），用假筛选法对血清中非特异性的抗噬菌体蛋白抗体和抗大肠杆菌抗体进行有效的吸附，以确保伪旋毛虫新生幼虫cDNA文库免疫学筛选的特异性。第一轮筛选到阳性克隆54个，以第一轮筛选结果为基础，进行第二轮筛选，最终得到阳性克隆10个，并对其进行测序和序列分析，并对其生物学特性进行相关阐述。在10个测序的基因中，有1条已知基因，将之命名为HW7,与ABL09499.2基因同源达到100%。有2条未知基因，将之命名为HW1，HW9。另外7条基因分别命名为HW1、HW2、HW4、HW5、 HW6、 HW8、 HW10：其中HW3与编码蛋白为神经胶细胞瘤病原相关蛋白-1(glioma pathogeneis-related protein1[Trichinella spiralis],XP_003378739.1)的同源性为84%;HW2与编码假定蛋白质(conserved hypothetical protein，ZP_06571682.1)同源性为36%；HW4与编码细胞色素C氧化酶I-型亚基(cytochrome c oxidase subunit I [Trichinella spiralis]，NP_077265.1)同源性为82%；HW5与编码II型脱氧核糖核酸酶超家族(deoxyribonuclease II superfamily [Trichinella spiralis],XP_003373067.1)同源性为81%,与新生幼虫期特异性DNaseII-12(newborn larvae-specific DNase II-12[T r i c h i n e l l a s p i r a l i s], A A X22752.1)同源性为79%; H W6抑制素(prohibitin[Trichinella spiralis], XP_003373863.1)同源性为96%; HW8与编码钙激活钾通道蛋白slo-1(calcium-activated potassium channel slo-1[Trichinellaspiralis],XP_003370272.1)同源性为93%; HW10与编码神经元钙传感蛋白质类-1(neuronal calcium sensor1[Trichinella spiralis],XP_003380590.1)同源性为92%。其中HW3具有完整的开放阅读框架(ORF),可编码蛋白的碱基序列最长。在筛选到的阳性克隆中，发现有期特异性HW5，编码蛋白与旋毛虫DNaseII超家族蛋白有很高的同源性，II型核酸酶在引发了肌细胞胶原蛋白基因的异常转录与表达，与伪旋毛虫体外形成了的薄胶原蛋白包囊有直接关系。目前本实验室已分别完成伪旋毛虫感染后26d、60d后抗血清对新生幼虫时期cDNA文库的的免疫学筛选，本论文成功使用感染后32d的抗血清对新生幼虫时期cDNA文库的免疫学筛选，并得到相关抗原性基因。该研究可进一步对所筛选到的目的基因采用原核表达系统在体外进行大量表达，建立敏感、特异的伪旋毛虫病的早期免疫学检测方法，以及为伪旋毛虫病的早期诊断和疫苗研发及功能基因的研究奠定基础。
[Abstract]:Trichinelliasis (trichinosis/trichinellosis) is a zoonotic foodborne parasitic disease caused by Trichinella infection, the distribution range is extremely wide, concentrated in less developed countries and some developed countries have the habit of hunting in Southeast Asia. In recent years the disease report is greatly improved, the reason may be with the improvement of living standards, people the demand for meat also increased, cross regional tourism behavior also caused the disease to spread. The distribution range in North America, Europe and Australia, beef, horse meat and other meat meat products is an important cause of human Trichinella infection. China in Northeast China, Guangxi Tibet, and Yunnan, reported the incidence caused by local eating habits have also occurred, causing serious social impact of social panic, which caused the It's extremely serious.
In Schistosoma japonicum have been reported in the twelve species, t. pseudospiralis is the only one that can infect mammals can also infect birds of Trichinella spiralis, greater mobility, difficult to prevent and control, is different from other kinds of special epidemic distribution and infection characteristics. Adult period lasted for 30d in the host gut the invasion time, newborn larvae produced a 4 month long muscle invasion period, and the whole muscle cells to infection, infection bring long lasting pain. The diagnosis, mainly direct muscle or muscle tabletting microscopy after digestion by optical microscope, due to Trichinella pseudospiralis infection period not formed cysts in the muscle or only the formation of cysts is thin, so the traditional method of microscopic examination of muscle compression is difficult to detect the pathogen; in addition due to the limitation of moral and ethical aspects of the muscle biopsy in the human body is also difficult to achieve.
T.pseudospiralis in different development stages of surface antigen and secretory antigen is different, can induce different degrees of immunity. As the host immune response is mainly aimed at other times, newborn larvae travel in the blood is the life cycle not only in the initial stage of development in the host cells are parasitic, an important role in regulating the immune response of the host parasite. During the period of pseudo Trichinella spiralis muscle larva survival in host muscle cells for several years or even longer, antibody production in vivo can be used for diagnosis. If the antibody in this period can also identify newborn larvae and adult period period worm antigen specific. You will find a gene for identifying worms in the entire life cycle. T.pseudospiralis used in this experiment (ISS13) of newborn larvae cDNA library, after the titer, determine its titer 4.25 * 107pfu/mL, totally accord with the standard. Before the immunoscreening of expression library, we must first amplification of nonspecific blue spots and determination of the titer of blue spots, to reach a certain magnitude after (109pfu/mL), with a false screening for serum non-specific phage antibody and protein effectively. The adsorption of antibodies against Escherichia coli, in order to ensure the specificity of screening the cDNA Library of immunology pseudo newborn larvae of Trichinella spiralis. The first round of screening to 54 positive clones, with the first round of screening based on the results of the second round of screening, finally obtained 10 positive clones, was sequenced and analyzed the related elaboration on and the biological characteristics. Among the 10 sequenced genes, 1 known genes, named HW7, homologous with ABL09499.2 gene to 100%. 2 unknown genes, named HW1, HW9. and other 7 genes Don't be named HW1, HW2, HW4, HW5, HW6, HW8, HW10: HW3 and encoding a protein of glial cell tumor pathogenesis related protein -1 (glioma pathogeneis-related protein1[Trichinella spiralis], XP_003378739.1) 84% homology with HW2; encoding putative proteins (conserved hypothetical protein, ZP_06571682.1) 36% homology with HW4; encoding cytochrome C oxidase subunit I- (cytochrome c oxidase subunit I [Trichinella spiralis], NP_077265.1) homology is 82%; HW5 and II encoding type deoxyribonuclease superfamily (deoxyribonuclease II superfamily [Trichinella spiralis], XP_003373067.1) 81% homology with newborn larvae stage specific DNaseII-12 (newborn larvae-specific DNase II-12[T r i c h i n e l l a s p I r a l i s] A A X22752.1), the homology was 79%; H W6 prohibitin[Trichinella spiralis (hormone inhibition ], XP_003373863.1) 96% homology with HW8 encoding; calcium activated potassium channel protein slo-1 (calcium-activated potassium channel slo-1[Trichinellaspiralis], XP_003370272.1) 93% homology with HW10 encoding; neuron calcium sensor protein -1 (neuronal calcium sensor1[Trichinella spiralis], XP_003380590.1) with homology to 92%. in which HW3 has a complete open reading frame (ORF). The nucleotide sequence encoding the protein can be long. In the positive clones screened, found to have stage specific HW5, encoding protein and DNaseII family protein of Trichinella spiralis have very high homology, II nuclease in triggering abnormal transcription of muscle cells and collagen gene expression, there is a direct relationship between thin collagen and cyst in vitro formation of Trichinella pseudospiralis.
At present, the laboratory of Trichinella pseudospiralis 26D after infection respectively, after 60d antiserum on newborn larvae cDNA library Immunoscreen the period, the successful use of antiserum after 32D infection on the immune period of newborn larvae cDNA library screening, and get the antigenicity of gene. The study can further target gene of the screened by the prokaryotic expression system for expression, established in vitro sensitive early immunological method to detect specific pseudo trichinosis, and lay the foundation for study on early diagnosis and vaccine development and functional genes for pseudo trichinosis.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

【参考文献】