【作者】 卢艳；

【导师】 王小宁；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 血吸虫病是一种严重危害人类健康和阻碍流行区社会经济发展的寄生虫病。诊断是确定感染的有效手段,在防治工作中处于重要位置。无论从个体水平确定药物化疗的对象、考核化疗的效果,还是在群体水平监测血吸虫病疫情的变化、考核血吸虫病防治效果和评估流行趋势等等,均需要以诊断结果作为评判依据之一。传统的病原学方法是诊断血吸虫病最为可靠和经典的途径,但费时、费力且敏感性不高,难以适应大规模现场防治的需求。免疫学诊断方法具有较高的敏感性、特异性和实用性等优点,在血吸虫病的监测、防治以及疗效考核等方面具有广泛的应用价值。诊断抗原的筛选和鉴定不仅是免疫学诊断研究的核心内容,并且是建立免疫学诊断方法的基础。本研究利用吡喹酮治疗前后日本血吸虫病人血清分别筛选成虫、虫卵的cDNA表达文库,寻找可用于日本血吸虫病检测或疗效考核的潜在靶点,初步筛选获得了120个阳性克隆。结合阳性克隆的反应强度、分类和插入片段的大小,将所获的17个阳性克隆的插入片段进行测序,对其DNA序列进行生物信息学分析。根据序列分析的结果,选择了10个目的基因片段进行表达,利用生物信息学软件分析编码蛋白的性质并预测其功能。以阳性克隆SJA4-6、SJA6-5、SJE18-4、SJE20-4、SJE22-1、SJE22-4、SJE28-2和SJE28-6为模板,成功构建了10个含有目的基因的重组质粒(GST tag),重组质粒转化大肠杆菌BL21菌株,经IPTG诱导后获得了9种融合表达的重组蛋白。其中5个重组蛋白含有可溶性表达组分,4个重组蛋白为包涵体。利用亲和层析的方法对含有重组蛋白的菌体裂解液进行纯化,获得了4种纯度较高的重组蛋白,分别为rSjP1、rSjP2、rSjWSC1P和rSjEFCAB。采用免疫学方法初步评估了所获重组蛋白的免疫诊断价值,以rSjP1、rSjWSC1P和rSjEFCAB为包被抗原的间接ELISA方法可明显区分日本血吸虫病人混合血清和正常人混合血清,病人血清反应的OD值(P)均达到正常人的(N)2.1倍以上,其中重组蛋白rSjEFCAB的P/N值达到3.9,表明该蛋白具有良好的应用前景。建立了以rSjWSC1P和rSjEFCAB作为包被抗原的间接ELISA方法。该方法检测日本血吸虫病人的敏感性分别为72%(36/50)、74%(37/50)；检测非流行区正常人的假阳性率为0-3.3%(0/30-1/30)；与并殖吸虫病人无交叉反应,与囊虫病人、旋毛虫病人的交叉反应率分别为10%(1/10)和11%(1/9),与华支睾吸虫病人的交叉反应率分别为0(0/5)和20%(1/5)。研究结果表明该方法具有较高的敏感性和特异性,重组蛋白rSjWSC1P和rSjEFCAB是日本血吸虫病潜在的诊断抗原。同时,本研究还采用免疫沉淀反应分离血吸虫病人血清中的循环抗原,利用质谱和生物信息学软件分析循环抗原的蛋白序列,寻找高丰度的循环抗原分子。鉴于禽类与哺乳类动物的遗传距离较远,来源于禽类的抗体不易发生交叉反应,本研究制备和纯化了抗血吸虫成虫抗原的卵黄抗体(IgY),并建立了基于免疫沉淀反应(以IgY抗体做为捕捉系统)和蛋白质组学技术鉴定日本血吸虫循环抗原的方法。应用该方法从日本血吸虫病人血清中富集循环抗原,鉴定出7种蛋白组分,为循环抗原的深入研究提供了新的途径；同时为发展基于IgY和循环抗原的血吸虫病早期诊断或疗效考核方法奠定了基础。本研究为日本血吸虫病检测或疗效考核诊断抗原的筛选、鉴定以及发展相应的诊断技术提供了新的科学依据。更多还原

【Abstract】 Schistosomiasis is a serious, poverty-related public health problem in endemic countries. Sensitive diagnosis is the effective way to determine infection which plays an important role in the improvement of control and prevention strategies for schistosomiasis. Moreover, some strategies are built on the results of diagnosis, such as individual and community treatment, monitoring the epidemic situation changes, assessment of chemotherapeutic interventions and evaluation of epidemic trend. Traditional parasitological method is a reliable and classical approach for diagnosis of schistosomiasis. However, it is labor-intensive and time-consuming along with low sensitivity which would result in under-estimation in prevalence and infection intensity, particularly in the areas with low prevalence or after intervention. Therefore, the test is not an ideal field method for large-scale control of schistosomiasis.Immunological diagnosis would yield a higher sensitivity, specificity and practicality. This method is applied widely to control and prevention the disease and to evaluate the chemotherapeutic effect. The study of diagnostic antigens is the core field of immunological diagnoses because most methods are based on a good antigen.In this study, the adult worm and egg cDNA libraries were immunoscreened with the pre-treament and post-treatment serum samples from schistosomiasis japonica patients to obtain promising targets for detection or assessing therapeutic efficacy. For the first round, 120 positive clones were screened from the cDNA libraries. According to response intensity, categories and the length of inserts, we selected 17 positive clones to sequence the inserted fragments. Then, the DNA sequences were analyzed by bioinformatics tools.Based on the analysis results,10 target genes were selected for cloning and expression. The proteins which encoded by the genes were predicted their structures and functions. Positive clones SJA4-6, SJA6-5, SJE18-4, SJE20-4, SJE22-1, SJE22-4, SJE28-2 and SJE28-6 were used as templates to amplify the gene segments and construct 10 recombinant plasmids successfully. The recombinant plasmids were transformed into E.coli BL21. After the induction by IPTG,9 fusion proteins were espressed. There were 5 recombinant proteins having the soluble components and 4 recombinant proteins forming inclusion bodies. Through the affinity chromatography,4 recombinant proteins were purified from the bacterial lysate. Finally, we obtained rSjP1, rSjP2, rSjWSClP and rSjEFCAB products with high purity. The diagnostic values of purified recombinant proteins were evaluated by immunologic assay. An indirect enzyme-linked immunosorbent assay (ELISA) which used rSjPl, rSjWSC1P and rSjEFCAB as coating antigen respectively, could distinguish the pooled sera of normal persons from the pooled sera of schistosomiasis japonica patients. The optical density value of patient sera was 2.2-3.9 times higher than normal sera. When used rSjEFCAB as coating antigen, the ratio of OD value of the patient sera and the normal sera (P/N) was 3.9. The results showed that recombinant protein rSjEFCAB had promising application foreground.The sensitivity for detecting patients infected with Schistosoma japonicum by the indirect ELISA which used rSjWSC1P and rSjEFCAB were 72% (36/50) and 74% (37/50), respectively. The false-positive rates of detecting sera from normal people were 0-3.3% (0/30-1/30). When testing sera from patients infected with Paragonimuspulmonanis, Cysticercus cellulosae, Trichinella spiralis and Clonorchis sinensis by this method, the cross reaction rate was not observed in paragonimiasis,10%(1/10) in cysticercosis,11%(1/9) in trichinosis and 0-20%(0/5-1/5) in clonorchiasis. The results proved that recombinant proteins rSjWSC1P and rSjEFCAB had comparative higher sensitivity and specificity in detecting schistosomiasis. These recombinant proteins may be potential diagnostic antigens for schistosomiasis japonica.We also used immunoprecipitation to isolate the circulating antigen (CA) from schistosomiasis japonica patients’ sera. Then the antigens were identified by LC-MS/MS and advanced bioinformatics tools to find high abundance antigen molecules. Because of the great genetic diversity between avian and mammalian animals, the antibodies from avian showed litte interaction with human’s. In this study, the egg yolk immunoglobulin (IgY) against adult worm antigens were prepared and purified from immunized egg yolk. We developed a novel method based on IgY for identification and profiling CA in serum of schistosomiasis patients’ sera. The IgYs against adult worm antigens were used as the capture antibodies to enrich the CA through immunoprecipitation, then the CA was determined by proteomics analysis. There were 7 proteins, including syntaxin 1 A, heterogeneous nuclear ribonucleoprotein H’, carbonyl reductase 1, leucine carboxyl methyltransferase 1, fatty acid binding protein variant A, SJCHGC05715 protein and SJCHGC06828 protein, were identified as the components of CA. This method would be a new approach for further study of CA and lay a foundation for early diagnosis and evaluation of chemotherapy based on IgY and CA. This study presented the profile of schistosome CA which is essential for further development of diagnostic reagents for schistosomiasis.In conclusion, this study has provided scientific basis for the screening and identification of the specific diagnostic antigens for detection or assessing therapeutic efficacy of schistosomiasis japonica, and for future development of the relative technique.更多还原