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  本文選題：丹毒絲菌　切入點：保護(hù)性抗原篩選　出處：《湖南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究從豬紅斑丹毒絲菌全基因組水平著手,選擇可編碼金屬轉(zhuǎn)運相關(guān)蛋白、表面蛋白、膜蛋白、脂蛋白的閱讀框進(jìn)行跨膜區(qū)域分析、信號肽分析后設(shè)計51對引物。利用PCR基因克隆技術(shù)對選取蛋白進(jìn)行基因克隆表達(dá),經(jīng)過PCR擴增、酶切、連接、轉(zhuǎn)化后用特異性引物檢測,結(jié)果顯示51個基因片段成功插入pET-28a(+)載體。將構(gòu)建好的重組質(zhì)粒轉(zhuǎn)入大腸桿菌BL21 (DE3)中進(jìn)行誘導(dǎo)表達(dá),經(jīng)SDS-PAGE電泳鑒定顯示有23個蛋白成功得到表達(dá),其中包括14個可溶性表達(dá)蛋白及9個包涵體表達(dá)蛋白。對23個蛋白進(jìn)行小鼠免疫保護(hù)試驗,最終顯示Su-1(即surface protective antigen SpaA)與丹毒絲菌商品苗免疫組的保護(hù)率一致,均達(dá)到100%保護(hù)；同時Surface proteinA(Spa)節(jié)段表達(dá)的Su-2-2蛋白與Su-2-4蛋白以及脂蛋白Lp-2蛋白免疫組小鼠也有17%的存活率,也顯示了一定的免疫保護(hù)效果。本實驗室對SpaA氨基端的免疫保護(hù)區(qū)域構(gòu)建重組SpaA-N蛋白并對其進(jìn)行了小鼠攻毒保護(hù)試驗,結(jié)果顯示了Spa-N蛋白對小鼠有100%的保護(hù)率。本研究以該重組蛋白制備A1(OH)3佐劑亞單位疫苗,并進(jìn)行豬的免疫攻毒試驗,對免疫豬進(jìn)行丹毒絲菌攻毒也顯示了100%的保護(hù)性,結(jié)果表明Spa-N蛋白丹毒絲菌亞單位疫苗候選蛋白具有一定的可行性。亞單位疫苗誘導(dǎo)機體產(chǎn)生的抗體為疫苗所含抗原對應(yīng)的抗體,而野毒感染產(chǎn)生的抗體比較復(fù)雜,因此可以建立一種評估方法用來鑒別診斷丹毒絲菌野毒感染抗體與SpaA-N免疫抗體,本研究選取10個可溶性表達(dá)且表達(dá)量高的蛋白作為候選包被抗原包被ELISA反應(yīng)板檢測丹毒絲菌感染豬血清,結(jié)果顯示自然感染丹毒絲菌豬兩周后采得血清就能與Fe-2蛋白包被ELISA抗原板產(chǎn)生較強的反應(yīng),且Fe-2蛋白包被ELISA抗原板與大腸桿菌、葡萄球菌、鏈球菌、副豬嗜血桿菌、巴氏桿菌、沙門氏菌等細(xì)菌制備的高免血清的特異性試驗顯示其有較好的特異性,最終選定對丹毒絲菌自然感染血清反應(yīng)值較好的Fe-2蛋白作為鑒別診斷丹毒絲菌野毒感染抗體與SpaA-N免疫抗體的包被抗原,通過實驗優(yōu)化ELISA條件為：抗原包被量100ng/孔,血清稀釋倍數(shù)1/100,血清作用時間、酶標(biāo)二抗作用時間均為30min,底物作用時間15min。
[Abstract]:In this study, the reading frame encoding metal-transport associated protein, surface protein, membrane protein and lipoprotein was selected from the whole genome level of erysipelas erythematosus for transmembrane region analysis. After the signal peptide analysis, 51 pairs of primers were designed. The selected protein was cloned and expressed by PCR gene cloning technique. The selected protein was amplified by PCR, digested by enzyme, ligated, transformed and detected by specific primers. The results showed that 51 gene fragments were successfully inserted into pET-28a () vector. The constructed recombinant plasmid was transferred into E. coli BL21 DE3 to induce expression, and 23 proteins were successfully expressed by SDS-PAGE electrophoresis. There were 14 soluble expression proteins and 9 inclusion body expression proteins. The protective rate of Su-1 (surface protective antigen SpaA) was the same as that of commercial vaccine of Rhizoma erysipelis, and the protective rate of Su-1 (surface protective antigen SpaA) was 100%. At the same time, the mice immunized with Su-2-2 protein, Su-2-4 protein and lipoprotein Lp-2 protein also had a survival rate of 17%. The recombinant SpaA-N protein was constructed in the immunological protection region of SpaA amino terminal and the mice were tested for the protection of the recombinant SpaA-N protein. The results showed that Spa-N protein had a protective rate of 100% to mice. In this study, the recombinant protein was used to prepare the A1OHH3 adjuvant subunit vaccine, and the porcine immunization test was carried out. The protective effect of the recombinant protein on immunized pigs was also shown to be 100%. The results showed that the candidate protein of Spa-N protein subunit vaccine was feasible. The antibody produced by subunit vaccine was the antibody corresponding to the antigen contained in the vaccine, but the antibody produced by wild virus infection was more complex. Therefore, an evaluation method can be established to differentiate the antibody against the field virus infection of Rhizoma erysipelas from the immune antibody of SpaA-N. In this study, 10 soluble and high expression proteins were selected as candidate envelope antigen coated ELISA reaction plates for detection of porcine serum infected with Rhizoma erysipelas. The results showed that the serum collected after two weeks of natural infection could react strongly with Fe-2 protein coated with ELISA antigen plate, and Fe-2 protein coated ELISA antigen plate with Escherichia coli, Staphylococcus, Streptococcus, Haemophilus parasuis, Pasteurella spp. The specificity test of high immunity serum prepared by Salmonella and other bacteria showed that it had good specificity. Finally, the Fe-2 protein with good serum response value to natural infection of Rhizoma erysipelas was selected as the coating antigen for differential diagnosis of the antibody against Rhizoma erysipelas and the immune antibody against SpaA-N. The optimal conditions of ELISA were as follows: the amount of antigen envelope was 100ng / well. The dilution times of serum 1 / 100, the time of serum action and the time of action of enzyme labeled second antibody were 30 min and 15 min respectively.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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