本文選題：日本血吸蟲　切入點(diǎn)：Sjc23-LED抗原　出處：《吉林大學(xué)》2011年碩士論文

【摘要】：血吸蟲病是世界范圍內(nèi)對(duì)人類健康威脅最嚴(yán)重的寄生蟲病之一,流行于76個(gè)國家和地區(qū),至少2億人及大量牲畜被其感染,受威脅人口逾7億。其中日本血吸蟲是血吸蟲中生物學(xué)特性最復(fù)雜、危害最嚴(yán)重、防治難度最大的一種,也是我國唯一一種感染人的血吸蟲。 相對(duì)于化療短期的治病效果,免疫可提供長期的保護(hù)力,因此疫苗的研制和應(yīng)用被認(rèn)為是控制疫病的根本手段之一。多年來,人們?cè)诳瓜x疫苗上進(jìn)行了大量嘗試,但效果均不理想。目前疫苗研究的方向,多為亞單位疫苗,其關(guān)鍵步驟是對(duì)血吸蟲保護(hù)性抗原的篩選,然而此工作一直進(jìn)展緩慢,其中一個(gè)重要原因是對(duì)蟲體的免疫逃避機(jī)制和參與宿主的免疫調(diào)節(jié)過程缺少認(rèn)識(shí)。 日本血吸蟲23kDa膜蛋白(Sjc23)是四跨膜蛋白(TSP)家族的一員,在蟲體生活史各階段的膜表面均有表達(dá),直接暴露于宿主的免疫系統(tǒng),是宿主對(duì)蟲體免疫應(yīng)答的首要靶標(biāo),是目前公認(rèn)的幾種具有潛力的候選疫苗和具有診斷價(jià)值的抗原分子之一。對(duì)血吸蟲膜相關(guān)蛋白的研究不僅可為疫苗候選抗原的篩選提供依據(jù),也對(duì)血吸蟲與宿主相互作用和疾病診斷的研究有著重要意義。 本研究以日本血吸蟲成蟲DNA為模板,PCR擴(kuò)增出編碼Sjc23大胞外區(qū)(Sjc23-LED)的基因,構(gòu)建表達(dá)載體pET-22b-Sjc23-LED,轉(zhuǎn)化基因工程菌BL21(DE3),誘導(dǎo)表達(dá)出含有6×His標(biāo)簽的可溶性蛋白,并用His GraviTrap非變性親和層析鎳柱純化出具有生物學(xué)活性的蛋白質(zhì)。 用Sjc23-LED與弗氏佐劑混合免疫BALB/c小鼠,發(fā)現(xiàn)其具有較強(qiáng)的免疫原性,誘導(dǎo)產(chǎn)生了高水平的抗體�？贵w分型結(jié)果表明免疫后小鼠血清中的抗體一直是IgG2a亞型,而自然感染小鼠雖然也一直以IgG2a抗體為主,但在感染后期出現(xiàn)了IgG1型抗體,且抗體效價(jià)隨時(shí)間的延長而逐漸升高。這表明Sjc23抗原激發(fā)產(chǎn)生以IgG2a為主的非細(xì)胞嗜性抗體,同時(shí)抑制機(jī)體對(duì)其它抗原的應(yīng)答,其自身可能是與血吸蟲免疫逃避相關(guān)的抗原分子。 鑒于正常患者血清與Sjc23有較強(qiáng)的粘附作用,本研究首先用ELISA法檢測(cè)人血清成分——IgA、IgE、IgG、IgM、白蛋白與Sjc23-LED的粘附情況,結(jié)果僅有IgG與Sjc23-LED具有親和力。為了進(jìn)一步驗(yàn)證二者結(jié)合的特異性,我們采用Pull-Down的方法,將重組蛋白固定在His GraviTrap磁珠上,構(gòu)成“誘餌”蛋白,捕獲可與其特異性結(jié)合的蛋白分子,進(jìn)一步用洗脫劑將蛋白復(fù)合物洗脫下來進(jìn)行Western Blot鑒定。結(jié)果依然是Sjc23-LED僅與非免疫人IgG具有親和力。該方法較ELISA受干擾因素少且要求配體-受體間具有更強(qiáng)的親和力,因此更具說服力。 日本血吸蟲是一種人獸共患寄生蟲,同樣也威脅著畜牧業(yè)的發(fā)展。因此我們對(duì)疫區(qū)常見家畜牛和豬的IgG與Sjc23-LED的親和力進(jìn)行了研究。ELISA和Pull-Down結(jié)果都顯示Sjc23-LED與豬IgG微弱結(jié)合、與牛IgG不結(jié)合,這表明Sjc23對(duì)宿主的免疫調(diào)節(jié)作用具有宿主差異性,血吸蟲對(duì)動(dòng)物機(jī)體的適應(yīng)程度可能要弱于人類。 為了對(duì)Sjc23-LED與非免疫人IgG相互作用的位點(diǎn)進(jìn)行定位,我們先用ELISA和Pull-Down的方法證明Sjc23-LED與Fc片段發(fā)生粘附。接著我們根據(jù)Sjc23-LED的氨基酸序列設(shè)計(jì)了7段重合肽,在Pull-Down實(shí)驗(yàn)中分別用這些肽段封閉人IgG的結(jié)合位點(diǎn)以抑制其與Sjc23-LED結(jié)合。結(jié)果肽段3、4顯示出抑制作用,即兩段肽段所共有的9氨基酸序列(-KIQTSFHCC-)介導(dǎo)了Sjc23-LED與人IgG的粘附。Sjc23-LED的三級(jí)結(jié)構(gòu)預(yù)測(cè)顯示,這9個(gè)氨基酸位于第二個(gè)螺旋,其后端保守而前端與家族其他成員不同,Sjc23-LED的三級(jí)結(jié)構(gòu)與CD81、SjcTSP-2的差異也標(biāo)志著其可能具有和其他TSP家族成員不同的生物功能。 本研究首次確定了血吸蟲配體表位和人類宿主受體之間的相互作用：Sjc23與宿主的免疫系統(tǒng)相互作用,迅速誘導(dǎo)產(chǎn)生低親和力的抗體,從而抑制宿主對(duì)其他抗原的免疫反應(yīng),是一個(gè)參與免疫逃避的調(diào)節(jié)抗原,可能并不適宜作為日本血吸蟲病的候選疫苗和診斷抗原分子。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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