【摘要】： 創(chuàng)傷弧菌(Vibrio vulnificus)是一種嗜鹽弧菌,廣泛存在于海水及海產(chǎn)品中,人體可通過生食海鮮或經(jīng)過肢體破損創(chuàng)口接觸海水、海產(chǎn)品而受其感染。常見于海上勞作的漁民及駐海演習人員從而引起敗血癥和軟組織感染。本菌對于糖尿病、酒精性肝病、肝硬化、肝炎、肝功能障礙患者的危害相當嚴重,可引起創(chuàng)傷弧菌膿毒血癥,起病急,進展迅速,死亡率高[1]�？焖贉蚀_的早期診斷是指導臨床正確用藥從而達到理想治療效果的有力保證;同時,創(chuàng)傷弧菌的治療手段仍為經(jīng)驗性的抗生素應用,然而,隨著細菌對抗生素逐漸出現(xiàn)的不同程度的耐藥現(xiàn)象,找到一種非大劑量應用抗生素的治療也成為亟待解決的問題。單克隆抗體用于疾病的診斷及治療已在醫(yī)學領(lǐng)域得到廣泛開展,并收到了很好的效果。作為我國海域主要致病菌之一的創(chuàng)傷弧菌,至今未見有診斷與治療的單克隆抗體制劑問世,為了解決該菌感染的診斷及防治問題,本研究對其單克隆抗體進行了研究。具體試驗方法如下: (1)通過動物免疫、細胞融合篩選克隆化及ELISA技術(shù)制備并鑒定抗創(chuàng)傷弧菌的單克隆抗體。獲得了2株穩(wěn)定分泌抗創(chuàng)傷弧菌的雜交瘤細胞株,命名為1A2(IgG1)和2D6 (IgG2a)。經(jīng)測定雜交瘤細胞培養(yǎng)上清及其誘生的腹水mAb效價分別為1×10-2 ,1×10-3和1×10-4 , 1×10-5。經(jīng)交叉試驗,2D6與溶藻弧菌、遲緩愛德華菌、鰻弧菌、溫和氣單胞菌以及嗜水氣單胞菌均不產(chǎn)生交叉反應,可以應用于創(chuàng)傷弧菌感染的快速檢測。 (2)以創(chuàng)傷弧菌分別感染乳鼠為模型,對1A2和2D6的治療保護效果進行研究。結(jié)果顯示: 1A2和2D6均能提高受創(chuàng)傷弧菌感染的乳鼠的存活率,分別為62.50%和37.50%,1A2具有明顯的保護作用。 本實驗通過雜交瘤技術(shù)共獲得2株分泌抗創(chuàng)傷弧菌單克隆抗體的雜交瘤細胞株1A2和2D6。2株雜交瘤細胞的培養(yǎng)上清及腹水能在較長時間內(nèi)基本保持穩(wěn)定。其中,1A2的培養(yǎng)上清、腹水效價及親和力均高于2D6;2D6與溶藻弧菌、遲緩愛德華氏菌、鰻弧菌、溫和氣單胞菌以及嗜水氣單胞菌均不產(chǎn)生交叉反應,可以應用于創(chuàng)傷弧菌感染的快速檢測。綜上,單克隆抗體2D6在快速檢測創(chuàng)傷弧菌感染方面具有重要意義,而1A2有望在治療方面得以進一步的研究。本實驗制備的抗創(chuàng)傷弧菌單克隆抗體為開展海上工作人員遇險后快速準確的檢測奠定了實驗基礎(chǔ),并且為進一步對于創(chuàng)傷弧菌感染治療的研究提供了理論依據(jù),不僅有著重要的實驗意義,并且具有較高的實用價值。
[Abstract]:Vibrio vulnificus (Vibrio vulnificus) is a halophilic vibrio, which is widely found in sea water and marine products. The human body can be infected by raw seafood or contact with sea water through broken limbs. Fishermen and drills who work at sea often cause septicemia and soft tissue infection. This bacterium is harmful to diabetes mellitus, alcoholic liver disease, liver cirrhosis, hepatitis and hepatic dysfunction. It can cause vibrio vulnificus sepsis, acute onset, rapid progress and high mortality [1]. Rapid and accurate early diagnosis is a powerful guarantee to guide clinical correct use of drugs so as to achieve an ideal therapeutic effect. At the same time, the treatment of Vibrio vulnificus is still an empirical application of antibiotics. However, with the increasing resistance of bacteria to antibiotics, finding a non-large dose treatment of antibiotics has become an urgent problem to be solved. Monoclonal antibodies have been widely used in the diagnosis and treatment of diseases. Vibrio vulnificus, one of the main pathogenic bacteria in the sea area of China, has not been found in the field of diagnosis and treatment. In order to solve the problem of diagnosis and prevention of the infection of Vibrio vulnificus, the monoclonal antibody of Vibrio vulnificus was studied in this study. The specific methods were as follows: (1) Monoclonal antibodies against Vibrio vulnificus were prepared and identified by animal immunization, cell fusion screening and ELISA. Two hybridoma cell lines stably secreting anti-Vibrio vulnificus were obtained and named as 1A2 (IgG1) and 2D6 (IgG2a). The mAb titers of hybridoma cell culture supernatant and ascites were 1 脳 10 ~ (2), 1 脳 10 ~ (- 3) and 1 脳 10 ~ (- 4), and 1 脳 10 ~ (- 5), respectively. 2D6 did not cross-react with Vibrio alginolyticus, Edelia tarda, Vibrio anguillarum, Aeromonas miliformis and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. (2) the therapeutic and protective effects of 1A2 and 2D6 in neonatal rats infected with Vibrio vulnificus were studied. The results showed that both 1A2 and 2D6 could increase the survival rate of newborn mice infected with Vibrio vulnificus by 62.50% and 37.50% respectively. 1A2 had obvious protective effect. In this study, two hybridoma cell lines 1A2 and 2D6.2 secreting monoclonal antibodies against Vibrio vulnificus were obtained by hybridoma technique. The supernatant and ascites of hybridoma cells were stable for a long time. Among them, the titer and affinity of 1A2 in culture supernatant were higher than 2D6; 2D6 did not cross-react with Vibrio alginolyticus, Edwards tarda, Vibrio eel, Aeromonas thermophilus and Aeromonas hydrophila, and could be used for rapid detection of Vibrio vulnificus infection. In summary, monoclonal antibody 2D6 plays an important role in rapid detection of Vibrio vulnificus infection, and 1A2 is expected to be further studied in the treatment of Vibrio vulnificus. The monoclonal antibodies against Vibrio vulnificus prepared in this experiment have laid an experimental foundation for rapid and accurate detection of Vibrio vulnificus after distress, and provided theoretical basis for further research on the treatment of Vibrio vulnificus infection. It not only has important experimental significance, but also has high practical value.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【引證文獻】

相關(guān)期刊論文 前1條

1 李曉莉;張以芳;曾令兵;許映芳;肖晶;童亮;;單克隆抗體在水產(chǎn)養(yǎng)殖中的應用[J];動物醫(yī)學進展;2009年09期

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本文編號：2466142