日本血吸虫23kDa膜蛋白与人免疫球蛋白的相互作用分析

发布时间：2018-03-25 17:42

本文选题：日本血吸虫　切入点：Sjc23-LED抗原　出处：《吉林大学》2011年硕士论文

【摘要】：血吸虫病是世界范围内对人类健康威胁最严重的寄生虫病之一,流行于76个国家和地区,至少2亿人及大量牲畜被其感染,受威胁人口逾7亿。其中日本血吸虫是血吸虫中生物学特性最复杂、危害最严重、防治难度最大的一种,也是我国唯一一种感染人的血吸虫。相对于化疗短期的治病效果,免疫可提供长期的保护力,因此疫苗的研制和应用被认为是控制疫病的根本手段之一。多年来,人们在抗虫疫苗上进行了大量尝试,但效果均不理想。目前疫苗研究的方向,多为亚单位疫苗,其关键步骤是对血吸虫保护性抗原的筛选,然而此工作一直进展缓慢,其中一个重要原因是对虫体的免疫逃避机制和参与宿主的免疫调节过程缺少认识。日本血吸虫23kDa膜蛋白(Sjc23)是四跨膜蛋白(TSP)家族的一员,在虫体生活史各阶段的膜表面均有表达,直接暴露于宿主的免疫系统,是宿主对虫体免疫应答的首要靶标,是目前公认的几种具有潜力的候选疫苗和具有诊断价值的抗原分子之一。对血吸虫膜相关蛋白的研究不仅可为疫苗候选抗原的筛选提供依据,也对血吸虫与宿主相互作用和疾病诊断的研究有着重要意义。本研究以日本血吸虫成虫DNA为模板,PCR扩增出编码Sjc23大胞外区(Sjc23-LED)的基因,构建表达载体pET-22b-Sjc23-LED,转化基因工程菌BL21(DE3),诱导表达出含有6×His标签的可溶性蛋白,并用His GraviTrap非变性亲和层析镍柱纯化出具有生物学活性的蛋白质。用Sjc23-LED与弗氏佐剂混合免疫BALB/c小鼠,发现其具有较强的免疫原性,诱导产生了高水平的抗体。抗体分型结果表明免疫后小鼠血清中的抗体一直是IgG2a亚型,而自然感染小鼠虽然也一直以IgG2a抗体为主,但在感染后期出现了IgG1型抗体,且抗体效价随时间的延长而逐渐升高。这表明Sjc23抗原激发产生以IgG2a为主的非细胞嗜性抗体,同时抑制机体对其它抗原的应答,其自身可能是与血吸虫免疫逃避相关的抗原分子。鉴于正常患者血清与Sjc23有较强的粘附作用,本研究首先用ELISA法检测人血清成分——IgA、IgE、IgG、IgM、白蛋白与Sjc23-LED的粘附情况,结果仅有IgG与Sjc23-LED具有亲和力。为了进一步验证二者结合的特异性,我们采用Pull-Down的方法,将重组蛋白固定在His GraviTrap磁珠上,构成“诱饵”蛋白,捕获可与其特异性结合的蛋白分子,进一步用洗脱剂将蛋白复合物洗脱下来进行Western Blot鉴定。结果依然是Sjc23-LED仅与非免疫人IgG具有亲和力。该方法较ELISA受干扰因素少且要求配体-受体间具有更强的亲和力,因此更具说服力。日本血吸虫是一种人兽共患寄生虫,同样也威胁着畜牧业的发展。因此我们对疫区常见家畜牛和猪的IgG与Sjc23-LED的亲和力进行了研究。ELISA和Pull-Down结果都显示Sjc23-LED与猪IgG微弱结合、与牛IgG不结合,这表明Sjc23对宿主的免疫调节作用具有宿主差异性,血吸虫对动物机体的适应程度可能要弱于人类。为了对Sjc23-LED与非免疫人IgG相互作用的位点进行定位,我们先用ELISA和Pull-Down的方法证明Sjc23-LED与Fc片段发生粘附。接着我们根据Sjc23-LED的氨基酸序列设计了7段重合肽,在Pull-Down实验中分别用这些肽段封闭人IgG的结合位点以抑制其与Sjc23-LED结合。结果肽段3、4显示出抑制作用,即两段肽段所共有的9氨基酸序列(-KIQTSFHCC-)介导了Sjc23-LED与人IgG的粘附。Sjc23-LED的三级结构预测显示,这9个氨基酸位于第二个螺旋,其后端保守而前端与家族其他成员不同,Sjc23-LED的三级结构与CD81、SjcTSP-2的差异也标志着其可能具有和其他TSP家族成员不同的生物功能。本研究首次确定了血吸虫配体表位和人类宿主受体之间的相互作用：Sjc23与宿主的免疫系统相互作用,迅速诱导产生低亲和力的抗体,从而抑制宿主对其他抗原的免疫反应,是一个参与免疫逃避的调节抗原,可能并不适宜作为日本血吸虫病的候选疫苗和诊断抗原分子。
[Abstract]:Schistosomiasis is one of parasitic diseases in the world the most serious threat to human health, popular in 76 countries and regions, at least 200 million people and a large number of animals infected, threatened a population of more than 700 million. The biological characteristics of Schistosoma japonicum in Schistosoma japonicum is the most complex, the most serious harm, prevention and treatment of a most difficult, but also I only in a human infection of Schistosoma japonicum.
Compared with the short-term curative effect of chemotherapy, immune force can provide long-term protection, so the development and application of the vaccine is considered to be one of the basic means to control the disease. Over the years, people have done a lot to try in insect vaccine, but the effect is not ideal. The current vaccine research direction for subunit vaccine, the key step on schistosome antigen screening protection, however, this work has been slow, one important reason is that the immune escape mechanism of insect host and participate in regulating process of lack of knowledge.
The 23kDa membrane protein of Schistosoma japonicum (Sjc23) is a transmembrane protein (TSP) family, the expression of membrane surface in each stage have insect life history, the immune system directly exposed to the host, the host is the primary target of insect immune response, is now recognized as one of several potential vaccine candidates and with the diagnostic value of the antigen molecule. Research on schistosome membrane associated protein can not only provide the basis for selection of vaccine candidate antigens, has important significance for schistosome host interactions and disease diagnosis.
In this study, adult Schistosoma japonicum DNA as template, amplified by PCR encoding Sjc23 extracellular domain (Sjc23-LED) gene and construct the expression vector pET-22b-Sjc23-LED, transforming gene engineering bacteria BL21 (DE3), induced expression of soluble protein containing 6 * His tag, and His GraviTrap non denaturing affinity chromatography with biological nickel column. The activity of the protein.
Sjc23-LED mixed with Freund's adjuvant immunized BALB/c mice, found that it has strong immunogenicity and induced high levels of antibodies. The antibody typing results showed that antibodies in sera of mice after immunization was IgG2a subtype, and naturally infected mice although also have been to IgG2a anti body, but appeared in the late stage of infection the IgG1 type antibody, antibody titer and extended with time gradually increased. This indicates that Sjc23 antigen induced heterophile antibody to IgG2a based non cells, while inhibiting the immune response to other antigens, the immune escape is possible with schistosome antigen molecules related.
In patients with normal serum and Sjc23 has strong adhesion, in this study, ELISA was used to detect the serum components IgA, IgE, IgG, IgM, adhesion of albumin and Sjc23-LED, the only IgG and Sjc23-LED have affinity. In order to verify the specificity of the combination of the two, we used the method of Pull-Down, the recombinant protein immobilized on His GraviTrap beads, a bait protein, can capture protein molecules and its specific binding, further eluted protein complexes were eluted with the Western Blot identification. The results is still only Sjc23-LED and IgG with non immune affinity. This method is ELISA and requires less disturbance the higher affinity ligand receptor, therefore more convincing.
Schistosoma japonicum is a parasitic zoonosis, also threaten the development of animal husbandry. So we in the affected area IgG and Sjc23-LED common cattle and pig affinity were studied with.ELISA and Pull-Down results showed that Sjc23-LED and IgG of porcine weak binding, no node with bovine IgG, suggesting that the immune regulatory effects of Sjc23 on the host the host has the difference, to the extent of Schistosoma japonicum on animal body is likely to be weaker than humans.
In order to Sjc23-LED and non IgG immune interaction site positioning, we first use the method of ELISA and Pull-Down showed that Sjc23-LED and Fc fragments adhesion. Then we designed according to the amino acid sequence of Sjc23-LED Peptide 7 coincidence in the Pull-Down experiment, respectively with the peptide binding site closed to inhibit the IgG and Sjc23-LED binding peptide 3,4. The results showed that the inhibitory effects of two peptides shared 9 amino acid sequence (-KIQTSFHCC-) mediates the adhesion of.Sjc23-LED and Sjc23-LED three level structure IgG forecast shows that these 9 amino acids in the second helix, the conservative front and back with other members of the family, three structure and CD81 Sjc23-LED SjcTSP-2, the difference also marks its may have different TSP family members and other biological functions.
This is the first study to determine the ligand interaction between Schistosoma form and human host receptors: the interaction between Sjc23 and the host immune system, antibody rapidly induced by low affinity, thereby inhibiting the host immune response to other antigens, is involved in regulation of immune escape antigen, may not be suitable as a vaccine candidate for schistosomiasis and diagnostic antigens.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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