本文選題：霍亂毒素B亞單位 + 霍亂毒素B亞單位和多角體的融合蛋白��； 參考：《浙江大學(xué)》2005年碩士論文

【摘要】：1. 霍亂毒素B亞基在家蠶桿狀病毒表達(dá)系統(tǒng)的表達(dá)與活性分析 霍亂毒素B亞基(cholera toxin B submit,CTB)是霍亂毒素的無毒單位,具有很 強(qiáng)的免疫原性,能刺激機(jī)體產(chǎn)生黏膜IgA和血清IgG抗體。B亞單位的主要功能是與 有核細(xì)胞上的GM1-神經(jīng)結(jié)苷脂結(jié)合而使得A亞單位進(jìn)入細(xì)胞,這個功能使它在免疫佐 劑、口服疫苗與多肽衍生疫苗的研究中具有越來越重要的作用。 在本論文中,霍亂毒素B亞單位與多角體蛋白的融合基因被克隆至昆蟲桿狀病毒 表達(dá)載體系統(tǒng)(BEVS)的轉(zhuǎn)移載體pBacPAK8中,使其置于多角體蛋白(polyhedrin) 基因啟動子控制之下,獲得重組轉(zhuǎn)移載體pBacPAK-mCTB。重組轉(zhuǎn)移載體DNA與經(jīng) Bsu36I酶切線性化的修飾型病毒Bm-BacPAK6 DNA共轉(zhuǎn)染家蠶BmN細(xì)胞,然后經(jīng)空 斑純化、PCR擴(kuò)增、Southern雜交等方法鑒定出含CTB基因的重組病毒 BacPAK-mCTB。將重組病毒BacPAK-mCTB感染BmN細(xì)胞及五齡幼蟲, SDS-PAGE 電泳分析顯示重組桿狀病毒的116kD的β-半乳糖苷酶條帶缺失,證實(shí)外源基因已成功 取代了LacZ基因:Western blotting分析發(fā)現(xiàn)在14kD及70kD處有分別都有明顯條帶。 其表達(dá)的融合蛋白產(chǎn)物經(jīng)ELISA檢測顯示細(xì)胞和蟲體表達(dá)分別為5.6μg/ml 54.4μg/ml.在蠶體中得到了高效表達(dá):而GM1-ELISA說明CTB五聚體的結(jié)合GM1- 神經(jīng)結(jié)苷脂的生物學(xué)功能。NOD小鼠的免疫增強(qiáng)實(shí)驗(yàn)則明顯的反映了CTB的免疫試劑 及佐劑功能:它能顯著增強(qiáng)胰島素對自身免疫疾病的治療效果,本實(shí)驗(yàn)誘導(dǎo)免疫耐受 的較佳組合為混合的100μg胰島素和10μg CTB含量的蠶血;而且單獨(dú)喂養(yǎng)CTB的 NOD小鼠也顯示出良好的免疫增強(qiáng)作用,能夠延緩NOD小鼠的糖尿病發(fā)作,制止進(jìn) ～步的胰島的惡化。 2. 小麥小孢子特異表達(dá)β-expansin基因TaEXPB2的克隆與表達(dá) Expansin是一種體外誘導(dǎo)分離的植物細(xì)胞壁伸展的蛋白,在修飾細(xì)胞壁基礎(chǔ)上使 細(xì)胞膨脹。Expansin的功能眾多,除了細(xì)胞生長,還包括營養(yǎng)生長、形態(tài)發(fā)生、授粉 受精、果實(shí)軟化等,并表現(xiàn)出高度組織、器官和細(xì)胞特異性。 本論文在小麥小孢子特異表達(dá)階段,經(jīng)基因組DNA提取、RT-PCR、Northern及 Southern技術(shù),首次鑒定出了一個小麥小孢子特異β-expansin cDNA-TaEXPB2
[Abstract]:1. Expression and activity Analysis of Cholera toxin B Subunit in Bombyx mori baculovirus expression system Cholera toxin B submitine CTB is a nontoxic unit of cholera toxin. Strong immunogenicity can stimulate the production of mucosal IgA and serum IgG antibody. B subunit the main function of the In nucleated cells, GM1-neurodatin binds to make subunit A enter the cell, a function that causes it to enter the immune system. Agents, oral vaccines and polypeptide derived vaccines play a more and more important role in the research. In this thesis, the fusion gene of cholera toxin B subunit and polyhedrosis protein was cloned into insect baculovirus. The transfer vector pBacPAK8 of the expression vector system BEVs was placed in polyhedrin). The recombinant transfer vector pBacPAK-mCTB was obtained under the control of gene promoter. Recombinant transfer vector DNA and meridian Bsu36I restriction linearized modified virus Bm-BacPAK6 DNA was co-transfected into silkworm BmN cells and then empty. Identification of Recombinant virus containing CTB Gene by Southern blotting BacPAK-mCTB. Infection of recombinant virus BacPAK-mCTB with BmN cells and fifth instar larva, SDS-PAGE Electrophoretic analysis showed the deletion of 尾 -galactosidase band in 116kD of recombinant baculovirus, which confirmed the success of exogenous gene. Western blotting analysis, which replaced the LacZ gene, showed that there were obvious bands in 14kD and 70kD. The expression of the fusion protein was 5.6 渭 g/ml by ELISA. 54.4 渭 g / ml. Highly expressed in silkworm bodies: GM1-ELISA indicates the binding of CTB pentamers to GM1- The biological function of dadoside. The immunoenhancement test of nod mice obviously reflects the immune reagents of CTB. And adjuvant function: it can significantly enhance the efficacy of insulin in the treatment of autoimmune diseases. This experiment induces immune tolerance. The best combination was 100 渭 g insulin and 10 渭 g CTB of silkworm blood, and fed with CTB alone. NOD mice also showed a good immune enhancement effect, which can delay the onset of diabetes in NOD mice and prevent the entry of diabetes mellitus. The deterioration of islets. 2. Cloning and expression of 尾 -expansin Gene TaEXPB2 in Wheat microspore Expansin is a kind of protein that induces the cell wall extension of plant in vitro. It is based on the modification of the cell wall. Expansin has many functions, including vegetative growth, morphogenesis, pollination, in addition to cell growth. Fertilization, fruit softening, etc., and showing high tissue, organ, and cell specificity. In this paper, the genomic DNA was extracted from wheat microspore specific expression stage. A wheat microspore specific 尾 -expansin cDNA-TaEXPB2 was first identified by Southern technique.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346;Q789

【引證文獻(xiàn)】

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1 荊贊革;蘿卜肉質(zhì)根根重性狀遺傳標(biāo)記分析與膨脹素基因家族的克隆[D];南京農(nóng)業(yè)大學(xué);2009年

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本文編號：1960137