本文選題：囊尾蚴病　切入點：六鉤蚴　出處：《蚌埠醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：(1)構(gòu)建包含豬帶絳蟲六鉤蚴期和囊尾蚴期特異性疫苗候選分子的囊尾蚴病多期復(fù)合基因疫苗。(2)評價該復(fù)合基因疫苗的免疫效應(yīng)。 方法：(1)分別提取豬帶絳蟲六鉤蚴和囊尾蚴總RNA，應(yīng)用RT-PCR方法分別擴增六鉤蚴期特異性抗原TSOL18和囊尾蚴期特異性抗原cC1編碼基因，另外設(shè)計引物應(yīng)用重疊延伸PCR技術(shù)通過一柔性接頭（linker）(Gly4Ser)3串聯(lián)這兩個基因，，使TSOL18基因在前，cC1基因在后，即融合基因TSOL18+linker+cC1（簡寫為TSOL18/L/cC1）。將TSOL18/L/cC1融合基因裝載至pMD18-T載體中，并測序驗證。提取pMD18-TSOL18/L/cC1質(zhì)粒DNA，經(jīng)Xho I和Nhe I雙酶切，純化后的酶切產(chǎn)物TSOL18/L/cC1基因通過T4連接酶與經(jīng)Xho I和Nhe I雙酶切并純化的pVAX1真核表達(dá)質(zhì)粒連接，構(gòu)建pVAX1-TSOL18/L/cC1重組質(zhì)粒，同時構(gòu)建pVAX1-TSOL18, pVAX1-cC1。將所構(gòu)建的重組質(zhì)粒通過陽離子聚合物介導(dǎo)轉(zhuǎn)染293T細(xì)胞，設(shè)轉(zhuǎn)染pVAX1空載體組和未轉(zhuǎn)染組轉(zhuǎn)染；轉(zhuǎn)染72h后分別收集細(xì)胞，提取轉(zhuǎn)染后的細(xì)胞總RNA，通過RT-PCR、間接免疫熒光、Western-blot鑒定融合基因在真核細(xì)胞中的表達(dá)狀態(tài)；以構(gòu)建的重組質(zhì)粒肌肉注射小鼠，以免疫組化檢測融合基因在小鼠骨骼肌的真核表達(dá)效果。（2）以pVAX1-TSOL18、pVAX1-cC1、pVAX1-TSOL18/L/cC1經(jīng)肌肉注射途徑免疫BALB/c小鼠，以流式細(xì)胞術(shù)檢測免疫后小鼠脾淋巴細(xì)胞胞內(nèi)細(xì)胞因子等細(xì)胞免疫指標(biāo)，并以間接ELISA法檢測相應(yīng)的特異性抗體及特異性抗體動態(tài)變化等體液免疫指標(biāo)。 結(jié)果：(1)構(gòu)建的pVAX1-TSOL18/L/cC1重組質(zhì)粒經(jīng)酶切電泳和測序結(jié)果與預(yù)期設(shè)計的基因序列一致。真核細(xì)胞轉(zhuǎn)染后通過RT-PCR檢測顯示除轉(zhuǎn)染pVAX1空載體組和未轉(zhuǎn)染組外，其余各組均有相應(yīng)mRNA的表達(dá)。真核細(xì)胞轉(zhuǎn)染后通過間接免疫熒光、Western-blot檢測表明能夠在293T細(xì)胞中表達(dá)出目的蛋白。免疫組化染色后在重組質(zhì)粒注射部位肌肉組織的肌纖維內(nèi)顯示棕黃色陽性分泌顆粒，并且pVAX1-TSOL18/L/cC1組比pVAX1-TSOL18組棕黃色陽性分泌顆粒多，空載體組與空白對照組骨骼肌組織未見明顯的棕黃色顆粒。（2）pVAX1-TSOL18/L/cC1組小鼠血清IgG抗體均顯著高于pVAX1-TSOL18、pVAX1-cC1組及PBS組和pVAX1組(P0.05)。 pVAX1-TSOL18/L/cC1組、pVAX1-TSOL18、pVAX1-cC1組分泌IFN-γ的CD4~+T及CD8~+T細(xì)胞各亞群比例均顯著高于PBS組和pVAX1組(P0.05)， pVAX1-TSOL18/L/cC1組又顯著高于pVAX1-TSOL18、pVAX1-cC1組(P0.05)；pVAX1-TSOL18/L/cC1組、pVAX1-TSOL18、pVAX1-cC1組分泌IL-4的CD4~+T細(xì)胞亞群比例顯著高于PBS組和pVAX1組(P0.05)，而pVAX1-TSOL18/L/cC1組、pVAX1-TSOL18、pVAX1-cC1組與PBS組和pVAX1組組間無顯著差異（P＞0.05）；pVAX1-TSOL18、pVAX1-cC1組分泌IL-4的CD8~+T細(xì)胞亞群比例顯著高于PBS組和pVAX1組(P0.05)，而pVAX1-TSOL18/L/cC1組與與PBS組和pVAX1組組間無顯著差異（P＞0.05）。 結(jié)論：（1）成功構(gòu)建包含豬帶絳蟲六鉤蚴和囊尾蚴期特異性疫苗候選分子的囊尾蚴病多期復(fù)合基因疫苗pVAX1-TSOL18/L/cC1。并且初步揭示了其在體內(nèi)和體外的表達(dá)。（2）囊尾蚴病多期復(fù)合基因疫苗pVAX1-TSOL18/L/cC1較單基因疫苗pVAX1-TSOL18、pVAX1-cC1誘導(dǎo)更為全面的免疫應(yīng)答效應(yīng)。
[Abstract]:Objective: (1) to construct cysticercosis multiphase complex gene vaccine containing six cysticercus cellulosae and cysticercus stage specific vaccine candidates. (2) evaluate the immune effect of the composite gene vaccine.
Methods: (1) were extracted from Taenia solium oncosphere and cysticercus six total RNA were amplified using RT-PCR method six oncosphere stage specific antigen TSOL18 and cysticercus stage specific antigen cC1 gene encoding, and primers were designed using overlap extension PCR technique through a flexible joint (linker) (Gly4Ser) of the 3 series two genes, the TSOL18 gene in the cC1 gene, after which the fusion gene TSOL18+linker+cC1 (TSOL18/L/cC1). The TSOL18/L/cC1 fusion gene loaded into pMD18-T vector, and pMD18-TSOL18/L/cC1 sequencing. Extraction of plasmid DNA by Xho I and Nhe I double enzyme digestion, pVAX1 eukaryotic purified enzyme digestion products TSOL18/L/cC1 T4 gene by I and Nhe by Xho ligase and I double enzyme digestion and purification of the expression plasmid to construct the recombinant plasmid of pVAX1-TSOL18/L/cC1, and construct pVAX1-TSOL18, recombinant plasmid pVAX1-cC1. constructed by cationic poly Compound mediated transfection of 293T cells with pVAX1 transfected with empty vector transfected group and untransfected group; after transfection of 72h cells were collected, extracted from the transfected cells total RNA by RT-PCR, indirect immunofluorescence, expression of Western-blot fusion gene identification in eukaryotic cells with the recombinant plasmid injected mice; construction and by immunohistochemical detection of fusion gene in skeletal muscle of mice with eukaryotic expression. (2) by pVAX1-TSOL18, pVAX1-cC1, pVAX1-TSOL18/L/cC1 intramuscular immunization of BALB/c mice, with the index of cellular immunity was detected by flow cytometry after immunization of mice spleen lymphocyte intracellular cytokines, and detected by indirect ELISA method corresponding the specific antibody and antibody dynamic changes of humoral immunity.
Results: (1) gene pVAX1-TSOL18/L/cC1 recombinant plasmid by restriction enzyme digestion and sequencing results with the expected design. After transfection of eukaryotic cells was detected by RT-PCR showed that the pVAX1 empty vector group and non transfected group, the expression of other groups have corresponding mRNA. After transfection of eukaryotic cells by indirect immunofluorescence. Western-blot detection showed that can express the target protein in 293T cells. The recombinant plasmid injection site muscle fibers in the muscle tissue after staining immunohistochemistry showed positive brown granules, and the pVAX1-TSOL18/L/cC1 group than in the pVAX1-TSOL18 group Brown positive granules, empty vector group and blank control group in muscle tissue was brown and yellow particles. (2) pVAX1-TSOL18/L/cC1 mice serum IgG antibody were significantly higher than that of pVAX1-TSOL18, pVAX1-cC1 group and PBS group and pVAX1 group (P0.05 pVAX1-TSOL18/L). /cC1 group, pVAX1-TSOL18 group, pVAX1-cC1 CD4~+T and IFN- secretion of CD8~+T cell subsets were significantly higher than that of PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group was significantly higher than that of pVAX1-TSOL18 group (P0.05, pVAX1-cC1); group pVAX1-TSOL18/L/cC1, pVAX1-TSOL18, CD4~+T cell subsets pVAX1-cC1 secretion of IL-4 was significantly higher than PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group, pVAX1-TSOL18 group and PBS group, pVAX1-cC1 group and pVAX1 group showed no significant difference (P > 0.05); pVAX1-TSOL18, the proportion of CD8~+T cell subpopulation group pVAX1-cC1 secretion of IL-4 was significantly higher than that of PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group and PBS group and pVAX1 there was no significant difference between groups (P > 0.05).
Conclusion: (1) the successful construction of cysticercosis contains multiple pVAX1-TSOL18/L/cC1. gene vaccine of Taenia solium oncosphere and cysticercus six stage specific vaccine candidate molecules and reveal its expression in vivo and in vitro. (2) cysticercosis and multiple gene vaccine pVAX1-TSOL18/L/cC1 than single gene pVAX1-TSOL18 vaccine, pVAX1-cC1 effect of immune response induction more comprehensively.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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