日本血吸虫表膜蛋白TSP的多态性分析和功能研究及电穿孔法介导恶性疟疾DNA疫苗与不同佐剂配伍的免疫效果比较

发布时间：2018-05-20 01:07

本文选题：日本血吸虫 + 四次跨膜蛋白　；参考：《北京协和医学院》2010年博士论文

【摘要】：血吸虫病严重威胁人类的健康,全球76个发展中国家仍有血吸虫病的流行,约有2亿血吸虫病患者。血吸虫体被(tegument)是虫体与宿主直接接触的表面,介导与宿主相互作用。对血吸虫表膜(tegument surface membrane)蛋白的研究,有助于了解血吸虫免疫逃避、免疫调节、营养摄取等机制,进而有可能发现新的干涉靶点。血吸虫体被存在着表达广泛且多样的四次跨膜蛋白家族(tetraspanin,TSP),曼氏血吸虫四次跨膜蛋白家族成员被认为是具有保护潜力的抗原分子,其中Sm-TSP-2更是获得高达～60%的保护力；而其在日本血吸虫中的同源分子Sj-TSP-2且存在广泛的多态性,与免疫逃避相关,提示这两个物种在进化过程中发生的歧化作用。因此,有必要对日本血吸虫的四次跨膜蛋白家族成员展开深入研究,希望找到针对日本血吸虫的新的抗原和药物靶点。本研究首先利用生物信息学方法在日本血吸虫中共鉴定了29个四次跨膜蛋白家族(Sj-TSP)成员。其中,除Sj-tsp-2和Sj-25外,新发现3个Sj-tsp基因存在不同程度的变异体；另外,2个Sj-tsp存在选择性剪接,提示可能参与血吸虫逃避宿主免疫反应的过程,不适合作为抗原候选分子。实时荧光定量PCR表明不同Sj-tsp基因在尾蚴、肺期童虫、雌雄成虫和虫卵中呈多态性转录；肺期童虫是对宿主免疫攻击最敏感的发育期,这一时期Sj-tsp基因的表达受到关注。其中,12种Sj-tsp基因在童虫的相对较高水平表达,(相对tubulin1),7种Sj-tsp基因的表达在尾蚴入侵宿主后呈显著增加,说明这7种分子对以适应宿主体内环境发挥一定的作用,但其中2种是变异基因分别是Sj-tsp-2和Sj25,另有1种为Sj-tsp-7存在选择性剪接,被认为是Sj-tsp基因既要在宿主体内发挥功能,又要逃避宿主攻击所采取的策略。体外对保守且童虫期表达的19个Sj-tsp基因成功进行RNAi,并探讨其对虫体的潜在影响。其中,4种Sj-tsp基因的沉默效率在80%以上,9种Sj-tsp基因的沉默效率在40%-60%之间。但以上Sj-tsp抑制以后,童虫的表膜完整性不受影响,且生存率与对照组相比无显著差异,提示Sj-tsp基因可能在体内发挥与宿主相互作用的功能,而体外则检测不到其抑制后对童虫的影响。综合以上结果,基于Sj-tsp家族成员的保守性,分期转录水平、大亲水环半胱氨酸数目等分子特征,我们初步筛选出5个具有潜力的Sj-tsp基因,为进一步的免疫保护试验奠定基础。疟疾是当今世界对人类危害最为严重的传染性疾病之一。全球每年有3亿-5亿人感染恶性疟,有100万-300万人因这一疾病而死亡,其中大多数为非洲撒哈拉以南地区5岁以下的儿童。近年来,由于耐药疟原虫株和耐药蚊媒的不断产生和扩散,使疟疾控制形势更为严峻。因此,研制安全有效的疟疾疫苗,成为控制疟疾的重要手段。本课题组前期工作借鉴了分子育种技术(即DNA改组)的随机重组原理,利用表位改组技术构建了多表位基因库,并用库免疫血清筛选出高抗原性的阳性克隆,发现其中VR312(改名为M.RCAg-1)基因克隆不仅能在小鼠模型诱导交叉免疫保护而且能在家兔模型中诱导抗恶性疟原虫抑制性抗体；D10基因克隆在家兔模型中诱导抗恶性疟原虫抑制性抗体。本实验通过采用优化的DNA真核表达载体、电穿孔的免疫途径,结合4-1 BBL和西咪替丁这两种佐剂等方面改进DNA疫苗的免疫效果。结果发现电穿孔的免疫途径在低剂量时即可诱导高水平的抗体滴度；pVAX1表达载体的免疫效果比VR1012表达载体较好,但4-1BBL和西咪替丁这两种佐剂均不能有效提高M.RCAg-1和D10 DNA疫苗单独免疫在小鼠体内诱导的抗体滴度。
[Abstract]:Schistosomiasis is a serious threat to human health, and there is still a epidemic of schistosomiasis in 76 developing countries around the world. There are about 200 million schistosomiasis patients. Schistosoma (tegument) is a surface that is a direct contact between the host and host, and mediates the interaction with the host. The study of the tegument surface membrane protein can help to understand the blood sucking. The mechanism of insect immune escape, immunomodulation, nutrition intake and so on, and then the possible discovery of new interference targets. Schistosoma bodies are present in a wide and diverse expression of the four transmembrane protein family (tetraspanin, TSP). Members of the four transmembrane protein family of Schistosoma mansoni are considered as protective potential antigen molecules, of which Sm-TSP-2 is obtained. The protection force of up to 60% is up to 60%, and its homologous molecule in Schistosoma japonicum has a wide variety of polymorphism, which is related to the immune escape, suggesting the disproportionation of the two species in the evolution process. Therefore, it is necessary to further study the four transmembrane protein family members of Schistosoma japonicum, hoping to find the target for Japan. The new antigen and drug target of Schistosoma.
In this study, 29 four transmembrane protein family (Sj-TSP) members of Schistosoma japonicum were identified by bioinformatics. In addition to Sj-tsp-2 and Sj-25, 3 Sj-tsp genes were found to have varying degrees of variant. In addition, 2 Sj-tsp had selective splicing, suggesting that it might be involved in Schistosoma to escape the host immune response. The process is not suitable as an antigen candidate.
Real time fluorescence quantitative PCR showed that different Sj-tsp genes were polymorphic in the cercariae, the lung stage children, the male and female adults and the eggs, and the lung stage was the most sensitive development period for the host immune attack, and the expression of the Sj-tsp gene was concerned at this time. Among them, 12 Sj-tsp genes were expressed at relatively high levels of the child, (relative tubulin1), and 7 species. The expression of Sj-tsp gene is significantly increased after the invasion of the cercariae, indicating that these 7 molecules play a role in adapting to the host environment, but 2 of them are Sj-tsp-2 and Sj25, respectively, and the other 1 are selective splicing for Sj-tsp-7. It is considered that the Sj-tsp gene is not only functioning in the host, but also escaping from the host. A strategy taken by a host attack.
In vitro, 19 Sj-tsp genes expressed in the conservative and child stage were successfully carried out with RNAi, and the potential effect on the insect body was explored. Among them, the silencing efficiency of the 4 Sj-tsp genes was more than 80% and the silence efficiency of the 9 Sj-tsp genes was between 40%-60%. There is no significant difference, suggesting that the Sj-tsp gene may play a function with the host in vivo, but in vitro it can not detect the effect of its inhibition on the child. Based on the conservatism of the members of the Sj-tsp family, the level of transcriptional transcription, the number of large hydrocysteine, we have preliminarily screened 5 of them. The Sj-tsp gene of the force lays the foundation for further immune protection test.
Malaria is one of the most serious infectious diseases in the world today. 300 million -5 million people worldwide are infected with falciparum malaria every year, and 1 million -300 million people die of this disease. Most of them are children under the age of 5 in sub Saharan Africa. In recent years, the emergence and proliferation of drug resistant strains of malaria parasites and resistant mosquito vectors have been developed in recent years. The malaria control situation is even more serious. Therefore, developing a safe and effective malaria vaccine has become an important means to control malaria.
In the previous work, we used the principle of random recombination of molecular breeding technology (DNA regrouping), and constructed a multi epitope gene pool using epitope modification technique, and screened the positive clones with high antigenicity with the immune sera of the library. It was found that VR312 (renamed M.RCAg-1) gene clones could not only induce cross immunization protection in mice model but also in mice model. It can induce anti Plasmodium falciparum antibody in rabbit model, and clone D10 gene in rabbit model to induce anti Plasmodium falciparum inhibitory antibody.
In this experiment, the immune effects of the DNA vaccine were improved by using the optimized DNA eukaryotic expression vector, the immune pathway of electroporation, combined with the two adjuvant such as 4-1 BBL and cimetidine. The results showed that the immune pathway of electroporation could induce the high level of antibody titer at low dose, and the immune effect of the pVAX1 expression vector was more than the VR1012 expression. The vectors were good, but the two adjuvants of 4-1BBL and cimetidine could not effectively enhance the antibody titer induced by M.RCAg-1 and D10 DNA vaccine alone.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【相似文献】