本文選題：日本血吸蟲 + 鈣網(wǎng)織蛋白��； 參考：《上海師范大學(xué)》2012年碩士論文

【摘要】：日本血吸蟲病仍然是流行于我國的人畜共患病之一，長期有效地控制病的流行有賴于高效安全疫苗的應(yīng)用。因此，開發(fā)高效、安全、實用的抗血吸蟲疫苗一直是血防研究中亟待解決的難題。大量研究表明：通過模擬輻照致弱血吸蟲尾蚴或童蟲疫苗（RAV）誘導(dǎo)的高保護性效應(yīng)機理來研發(fā)高效而又安全的分子疫苗，可能是突破上述難題的有效策略之一。因此，探索RAV的高保護性效應(yīng)機制成為研究者們關(guān)注的熱點課題，其中，RAV的免疫原性的分子與細胞基礎(chǔ)及其機制等問題尤其受到重視。 以前的研究還顯示：鈣網(wǎng)織蛋白（Calreticulin，CRT）是一類進化上較為保守的分子，由保守的球狀N端結(jié)構(gòu)域和脯氨酸富集結(jié)構(gòu)域、以及酸性的C端結(jié)構(gòu)域所組成，屬于Ca2+結(jié)合伴侶蛋白，最初定位于內(nèi)質(zhì)網(wǎng)，參與調(diào)節(jié)蛋白質(zhì)的正確折疊、基因轉(zhuǎn)錄和翻譯后修飾等過程。最近報道，經(jīng)某些藥物或紫外照射等處理的腫瘤細胞以及凋亡的細胞的CRT會移位至細胞表面，從而給巨噬細胞和樹突細胞（Dendritic cells, DC）等抗原提呈細胞提供了一種“eat me”信號，最終導(dǎo)致這些細胞被吞噬和處理；同時，在這些細胞的其他應(yīng)急分子的共同作用下，還可增強腫瘤抗原的免疫原性。提示細胞表面CRT的暴露能夠引起其被DC吞噬，從而誘導(dǎo)更強的免疫反應(yīng)。另外，Naglaa El Gengehi等人鑒定了曼氏血吸蟲的CRT為輻照致弱疫苗的一種免疫顯性T細胞抗原，本課題組陳雷等也發(fā)現(xiàn)日本血吸蟲的CRT（SjCRT）含有一個雜合型的Th1細胞表位，說明血吸蟲的CRT可能是血吸蟲RAV中的一個重要的T細胞抗原。因此，可以推斷，在RA血吸蟲疫苗模型中，來源于血吸蟲的一些保守的應(yīng)急分子如CRT和HSP70等分子可能起到了關(guān)鍵作用，它們很可能通過與宿主DC作用，形成引發(fā)和驅(qū)使保護性的先天和適應(yīng)性免疫應(yīng)答的環(huán)境。但是，血吸蟲的CRT分子是否能夠活化宿主的DC誘導(dǎo)特征性的先天免疫反應(yīng)，仍有待進一步研究。因此，，本研究在克隆表達日本血吸蟲CRT基礎(chǔ)上，觀察了該分子在血吸蟲不同發(fā)育階段蟲體中的定位情況，并初步分析了SjCRT刺激小鼠抗原提呈細胞的功能特征，旨在為進一步闡明SjCRT在RAV中的免疫生物學(xué)功能和機制提供初步的資料。 首先，應(yīng)用常規(guī)分子生物學(xué)技術(shù)克隆SjCRT分子，并在原核表達系統(tǒng)中表達該分子，Western bloting分析了該蛋白分子的免疫原性，免疫熒光法觀察SjCRT分子在不同期別蟲體中的組織定位。結(jié)果克隆和表達了日本血吸蟲重組蛋白(rSjCRT)；Westen Blot分析顯示，該重組蛋白免疫鼠血清可以識別7d、14d、23d、32d和42d的日本血吸蟲的蟲體蛋白；免疫熒光檢測表明在7d、10d、14d日本血吸蟲童蟲體表膜呈現(xiàn)明顯的熒光信號。 為避免原核表達的蛋白含有脂多糖（LPS）而影響后續(xù)實驗，采用Bac-To-Bac桿狀病毒真核表達系統(tǒng)進行SjCRT蛋白的表達；His柱純化表達蛋白，Westen Blot分析蛋白免疫原性。結(jié)果在桿狀病毒表達系統(tǒng)中表達了SjCRT蛋白；Westen Blot分析顯示，純化后的SjCRT蛋白能夠被標簽His單抗識別，也能被原核表達SjCRT蛋白的免疫鼠血清所識別，為后續(xù)活化DC功能的實驗研究提供了材料。 RAW264.7巨噬細胞株具有較強的抗原吞噬功能，是常用的研究先天免疫功能的細胞模型。本實驗采用以原核表達后除去LPS的SjCRT蛋白作為抗原觀察其活化RAW264.7細胞的功能。CCK-8法檢測SjCRT蛋白刺激RAW264.7細胞的增殖情況；流式細胞術(shù)檢測SjCRT蛋白刺激后RAW264.7細胞表面標志分子MHCⅡ和TLR2與TLR4等受體分子的表達情況。結(jié)果表明，SjCRT蛋白能顯著地刺激RAW264.7細胞的增殖(SI2)； SjCRT蛋白刺激后實驗組RAW264.7細胞的MHCⅡ、TLR2和TLR4表達量顯著地增強(P0.05)。提示SjCRT蛋白能夠刺激RAW264.7細胞表型的成熟，且可能與TLR2和TLR4受體介導(dǎo)的過程相關(guān)。 為了解SjCRT是否具有活化宿主樹突狀細胞的功能，本研究采用上述真核表達的SjCRT蛋白刺激小鼠骨髓里來源的DC細胞，分析刺激后DC表型和功能成熟的情況。常規(guī)方法從Balb/c小鼠骨髓分離、培養(yǎng)和分選得到純度較高的不成熟的DC細胞；流式細胞術(shù)檢測SjCRT蛋白刺激DC細胞表面標志分子MHCⅡ、CD40和CD86的表達；ELISA法檢測刺激的DC細胞培養(yǎng)上清中的細胞因子IFN-γ、IL-10、TNF-α和IL-6的含量。結(jié)果顯示，SjCRT蛋白刺激后MHCⅡ和CD86表達顯著性增強(P0.05)；DC細胞分泌到上清中的IFN-γ顯著性增多(P0.05)，IL-10、TNF-α和IL-6顯著性減少(P0.05)。提示SjCRT蛋白能夠刺激小鼠骨髓來源的DC細胞表型的成熟，且具有促進DC細胞分泌Th1類致炎細胞因子IFN-γ的功能。 總之，本研究克隆表達了日本血吸蟲CRT分子，觀察到該分子能夠在7d、10d、14d日本血吸蟲童蟲體表膜呈現(xiàn)，初步查明SjCRT蛋白能夠活化宿主的樹突狀細胞和巨噬細胞，為進一步闡明SjCRT在RAV中的免疫生物學(xué)功能和機制奠定了基礎(chǔ)。
[Abstract]:As a result , the high protective effect mechanism of RAV - induced high protective effect is one of the most effective strategies to solve the problem . Therefore , the research of RAV ' s high protective effect mechanism has become one of the most important issues of the researchers .

Previous studies have shown that Calreticulin ( CRT ) is a kind of evolutionarily conserved molecule , composed of conserved globular N - terminal domain and proline - rich domain , and acidic C - terminal domain . It is composed of conserved globular N - terminal domain and proline - rich domain , and acidic C - terminal domain . It is initially located in the endoplasmic reticulum , participates in regulating the correct folding of protein , gene transcription and post - translational modification . Recently , it has been reported that tumor cells treated with certain drugs or ultraviolet irradiation and the CRT of apoptotic cells can be shifted to the surface of the cell , thus providing a " eat me " signal to the antigen - presenting cells such as macrophages and dendritic cells ( DC ) , which eventually lead to the phagocytosis and treatment of these cells ;
In addition , Naglaa El Gengehi et al . have identified that the CRT of Schistosoma japonicum may be an important T - cell antigen in RAV of Schistosoma japonicum . In addition , Naglaa El Gengehi et al . have identified the localization of some conserved emergency molecules such as CRT and HSP70 in Schistosoma japonicum .

Firstly , SjCRT molecule was cloned by conventional molecular biology technique and expressed in prokaryotic expression system . Western bloting analyzed the immunogenicity of the protein molecule . Immunofluorescence method was used to observe the localization of SjCRT molecule in the non - synchronous insect body .
Westen Blot analysis showed that the recombinant protein immune mouse serum can recognize the worm protein of Schistosoma japonicum at 7 , 14 , 23d , 32d and 42 days .
Immunofluorescence detection showed that the body surface membrane of Schistosoma japonicum showed obvious fluorescence signal at 7d , 10d and 14d .

In order to avoid the effect of LPS on the expression of SjCRT protein in prokaryotic expression system , the expression of SjCRT protein was carried out in the eukaryotic expression system .
The results showed that SjCRT protein was expressed in baculovirus expression system .
Western blot analysis showed that SjCRT protein after purification can be identified by tag His monoclonal antibody , and can be recognized by the immune mouse serum of SjCRT protein expressed by prokaryotic expression , which provides the material for experimental research on subsequent activation of DC function .

RAW264.7 macrophage strain has a strong antigen - phagocytic function and is a common cellular model to study innate immune function . SjCRT protein is used as antigen to observe the function of activated RAW264.7 cells . CCK - 8 is used to detect the proliferation of RAW264.7 cells stimulated by SjCRT protein .
The expression of MHC 鈪

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