本文選題：番茄潰瘍病菌　切入點(diǎn)：多克隆抗體　出處：《蘭州大學(xué)》2011年碩士論文

【摘要】：目的：番茄潰瘍病(Bacterial canker of tomato)是一種嚴(yán)重危害番茄生產(chǎn)的細(xì)菌性病害,普遍發(fā)生在世界各大番茄主要產(chǎn)區(qū)。番茄潰瘍病的致病菌是密執(zhí)安棒桿菌(Clavibacter michiganensis subsp.michiganensis, Cmm)。Cmm主要是通過浸染番茄植株維管束系統(tǒng)從而導(dǎo)致植株干枯致死,由于Cmm具有致病性強(qiáng)、潛伏期長、易于傳播等特點(diǎn),對(duì)番茄潰瘍病的產(chǎn)量和質(zhì)量造成了嚴(yán)重?fù)p失帶來了巨大的經(jīng)濟(jì)損失。Cmm病菌的檢測對(duì)于番茄潰瘍病的防治是至關(guān)重要的,番茄潰瘍病菌的檢測方法主要有生物學(xué)、血清學(xué)、分子生物學(xué)等方法,由于血清學(xué)檢測如酶聯(lián)免疫方法特異性好、靈敏度高且操作簡單而被廣泛應(yīng)用。但是目前國內(nèi)的番茄潰瘍病菌抗體全部依賴國外進(jìn)口,檢測成本較高,使得免疫學(xué)方法無法在國內(nèi)普及運(yùn)用。為此,我們制備了效價(jià)高、特異性好的多克隆抗體和單克隆抗體,其目的是逐漸解決番茄潰瘍病菌抗體和酶聯(lián)免疫試劑盒的國產(chǎn)化問題,打破國外公司在該領(lǐng)域的技術(shù)和產(chǎn)品壟斷,降低國內(nèi)該病菌檢測成本,促進(jìn)該類試劑盒在我國的普及運(yùn)用。 方法：用番茄潰瘍病菌全菌皮下免疫新西蘭兔, ELISA測抗體效價(jià),頸動(dòng)脈采血,收集血清,對(duì)抗體特異性鑒定,最后對(duì)制備的多克隆抗體進(jìn)行純化。用番茄潰瘍病菌全菌皮下免疫BALB/c小鼠制備單克隆抗體,采用B細(xì)胞雜交瘤技術(shù),經(jīng)免疫、融合、間接ELISA篩選和克隆等,獲得穩(wěn)定分泌抗體的陽性雜交瘤細(xì)胞株,最后對(duì)得到的番茄潰瘍病菌的單抗進(jìn)行效價(jià)、特異性、親和力等鑒定。 結(jié)果：制備了針對(duì)番茄潰瘍菌的多克隆抗血清,抗體效價(jià)為1：256000,Western Blot分析表明該抗體能特異性識(shí)別番茄潰瘍病菌；經(jīng)免疫BALB/c小鼠后獲得3株番茄潰瘍病菌單抗分別為1A4、1C3和1B7,經(jīng)亞類鑒定分別是IgM、IgG1、IgG1;純化腹水間接ELISA效價(jià)分別為1：25600、1：12800、1：12800；與其同屬不同亞種無明顯交叉反應(yīng)。 結(jié)論：制備的番茄潰瘍病菌多克隆抗體和3株單克隆抗體均具有較高特異性和敏感性,可作為番茄潰瘍病菌的檢測抗體,其中,1A4的特異性最好。番茄潰瘍病菌多克隆抗體和單克隆抗體的獲得為進(jìn)一步研發(fā)番茄潰瘍病檢測試劑盒奠定了基礎(chǔ)。
[Abstract]:Objective: the bacterial canker of tomato canker is a kind of bacterial disease which seriously endangers tomato production.The pathogen of tomato canker is Clavibacter michiganensis subsp. michiganensis. Cmm).Cmm is mainly caused by soaking the vascular bundle system of tomato plant to cause the plant to dry to death. Because Cmm has the characteristics of strong pathogenicity, long incubation period, easy to spread, etc.It is very important for the prevention and cure of tomato canker to detect the pathogen of Cmm, which has caused serious loss to the yield and quality of tomato canker. The detection methods of tomato ulcer pathogen mainly include biology and serology.Molecular biology and other methods have been widely used for serological detection such as enzyme linked immunosorbent assay (Elisa) because of its high specificity high sensitivity and simple operation.However, at present, the domestic antibodies against tomato ulcer bacteria all rely on foreign imports, and the cost of detection is high, which makes immunological methods can not be widely used in China.Therefore, we have prepared high titer and good specificity polyclonal antibodies and monoclonal antibodies, the purpose of which is to gradually solve the problem of localization of tomato ulcer pathogen antibodies and enzyme linked immunosorbent kits.To break the monopoly of technology and products of foreign companies in this field, to reduce the cost of detection of this pathogen in China, and to promote the popularization and application of this kind of kit in our country.Methods: new Zealand rabbits were immunized subcutaneously with tomato ulcer bacteria. The antibody titers were measured by ELISA, blood samples were collected from carotid artery, serum was collected, antibody specificity was identified, and the polyclonal antibody was purified.BALB/c mice were immunized subcutaneously with tomato ulcer bacteria to prepare monoclonal antibody. By using B cell hybridoma technique, immunizing, fusion, indirect ELISA screening and cloning, positive hybridoma cell lines secreting antibodies stably were obtained.Finally, the titer, specificity and affinity of the McAb of tomato ulcer pathogen were identified.Results: the polyclonal antiserum against tomato ulcer bacteria was prepared. The titer of the antibody was 1: 256 000. Western Blot analysis showed that the antibody could specifically identify tomato ulcer bacteria.After immunizing BALB/c mice, three McAbs of tomato ulcer pathogen were obtained, which were 1A4C3 and 1B7, respectively, and the titers of purified indirect ELISA from ascites were 1: 25600, 1: 12800 and 112800, respectively, and there was no obvious cross reaction with the same subspecies.Conclusion: the polyclonal antibody and three monoclonal antibodies of tomato ulcer bacteria have high specificity and sensitivity, and can be used as the detection antibody of tomato ulcer bacteria, among which the specificity of 1 A4 is the best.The acquisition of polyclonal antibodies and monoclonal antibodies against tomato ulcer bacteria laid a foundation for further research and development of tomato canker detection kit.
【學(xué)位授予單位】：蘭州大學(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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