本文選題：中試生產(chǎn) + DNA疫苗��； 參考：《武漢生物制品研究所》2010年碩士論文

【摘要】： DNA疫苗是近年來基因治療研究中所衍生并發(fā)展起來的一個新的研究領(lǐng)域。它是指將編碼某種蛋白質(zhì)抗原的重組真核表達載體免疫機體,使外源基因在活體內(nèi)表達,從而刺激免疫系統(tǒng)產(chǎn)生特異性的免疫應(yīng)答。本論文以我所中試生產(chǎn)的防齲DNA疫苗和結(jié)核DNA疫苗為基礎(chǔ),對這兩種疫苗免疫機體后誘導(dǎo)的免疫效能進行評價。 實驗一中試生產(chǎn)的防齲DNA疫苗效力評價試驗 本實驗以武漢大學口腔醫(yī)學院構(gòu)建的靶向融合防齲DNA疫苗pGJA-P/VAX1為基礎(chǔ)免疫定菌大鼠,研究靶向融合防齲DNA疫苗pGJA- P/VAX1的免疫防齲效果。將56只大鼠隨機分為7組,接種變形鏈球菌并給予致齲飼料,按3因素(質(zhì)粒,蛋白,佐劑)2水平(肌肉,粘膜免疫途徑)析因設(shè)計方案免疫大鼠,分別于第0、2、4、6、8、10周采集大鼠血清和唾液樣本。并建立大鼠血清IgG和唾液IgA的間接ELISA檢測方法,對建立的方法進行方法學驗證。完成了各組血清和唾液樣本的檢測。實驗結(jié)束時處死大鼠,參照Keyes經(jīng)典計分標準進行齲齒計分。SPSS軟件 分析結(jié)果表明: (1)疫苗免疫組特異性抗PAc血清IgG和唾液IgA抗體水平均顯著高于空載體免疫組(P0.0 1),而齲齒記分顯著低于空載體免疫組(P0.01 )。 (2)同一免疫途徑中,DNA疫苗初免、蛋白加強組特異性抗PAc血清IgG水平顯著高于單純DNA疫苗免疫組(P0.01),特異性抗PAc唾液IgA抗體水平、齲齒計分無顯著差異。 (3)同一免疫方案中,黏膜免疫組特異性抗PAc唾液IgA抗體水平高于肌肉免疫組,齲齒計分低于肌肉免疫組(P0.05),血清抗體水平無顯著差異 (4)黏膜免疫中,佐劑蛋白加強組的血清IgG和唾液IgA最高,顯著高于其他組(P0.01),齲齒計分單純DNA不加佐劑組高于其他三組(P0.05)。 由此得出結(jié)論:重組基因質(zhì)粒pGJA-P能有效的抑制齲病的發(fā)生和發(fā)展。滴鼻法和肌肉注射法接種防齲DNA疫苗都可有效防齲,而滴鼻法較肌肉注射法能更有效的控制齲損的發(fā)生,降低齲壞程度。 實驗二中試生產(chǎn)的結(jié)核DNA疫苗初步效力試驗 以解放軍309醫(yī)院構(gòu)建的編碼Ag85A抗原的結(jié)核DNA疫苗為基礎(chǔ)免疫小鼠,研究中試生產(chǎn)的TB DNA疫苗免疫效果。將4-6周齡的Balb/c小鼠20只隨機分為2組,一組免疫TB DNA疫苗,另一組免疫PBS作為陰性對照,于免疫前采血,共免疫三次,在第三次免疫后3周摘眼球處死小鼠,收集全血,分離血清,進行血清特異性抗體檢測和細胞因子(Interferon-γ、IL-4)檢測;同時對每只小鼠取脾細胞進行特異性Interferon-γ分泌檢測和脾細胞CD4+、CD8+百分率檢測。結(jié)果統(tǒng)計分析顯示: (1)TB DNA疫苗不能刺激小鼠機體產(chǎn)生顯著的特異性抗體。 (2)疫苗免疫組血清細胞因子IFN-γ水平顯著高于PBS對照組,而IL-4水平與PBS對照組之間無顯著差異。 (3)酶聯(lián)免疫斑點檢測結(jié)果表明TB免疫組脾細胞特異性IFN-γ分泌水平高于PBS組(P0.05)。 (4)TB DNA疫苗免疫組CD4+細胞百分率高于PBS對照組有統(tǒng)計學差異(0.025P0.05); CD8+細胞百分率與PBS對照組沒有統(tǒng)計學差異(0.2P0.25)。 綜合以上結(jié)果可以看出,TB DNA疫苗主要刺激機體Th1型細胞免疫,而對誘導(dǎo)機體產(chǎn)生Th2型細胞免疫應(yīng)答沒有顯著的促進作用。
[Abstract]:DNA vaccine is a new research field derived and developed in the research of gene therapy in recent years . It refers to the recombinant eukaryotic expression vector which encodes a certain protein antigen into the organism to stimulate the immune system to produce specific immune response .



Evaluation of Efficacy of Anti - caries DNA Vaccine for Trial Production



The anti - caries effect of anti - caries DNA vaccine pGJA - P / VAX1 was studied by targeting fused anti - caries DNA vaccine , pGJA - P / VAX1 , which was constructed by Wuhan University ' s oral medical school .



The analysis results show that :



( 1 ) The specific anti - PAc serum IgG and IgA antibody levels in the vaccinated group were significantly higher than that in the empty vector immune group ( P0.01 ) , but the caries score was significantly lower than that in the empty vector immune group ( P0.01 ) .



( 2 ) In the same immunization route , the serum IgG level of the DNA vaccine was significantly higher than that in the simple DNA vaccine group ( P0.01 ) .



( 3 ) In the same immunization protocol , the specific anti - PAc saliva IgA antibody level in the mucosal immune group was higher than that in the muscle immune group and the caries score was lower than that in the muscle immune group ( P0.05 ) .



( 4 ) In mucosal immunity , the highest levels of serum IgG and saliva IgA in adjuvant protein - enhanced group were higher than those in other groups ( P0.01 ) .



It is concluded that the recombinant plasmid pGJA - P can effectively inhibit the occurrence and development of dental caries . Both nasal drop method and intramuscular injection method can effectively prevent caries , and the nasal drop method can effectively control the occurrence of caries and reduce the degree of caries .



Preliminary efficacy test of tuberculosis DNA vaccine for trial production in experiment 2



Balb / c mice 4 - 6 weeks old were randomly divided into 2 groups , one group of immune TB DNA vaccine and one group of immune PBS as negative control , the whole blood was collected , serum was isolated , serum specific antibody detection and cytokines ( IL - 緯 , IL - 4 ) were collected .



( 1 ) TB DNA vaccine could not stimulate mouse body to produce significant specific antibody .



( 2 ) The level of IFN - 緯 in the serum of the vaccine group was significantly higher than that in the PBS control group , but there was no significant difference between the level of IL - 4 and the PBS control group .



( 3 ) ELISA results showed that the level of IFN - 緯 secretion in spleen cells of TB immunized group was higher than that in PBS group ( P0.05 ) .



( 4 ) The percentage of CD4 + cells in immunized group of TB DNA vaccine was higher than that in PBS control group ( 0.025 P0.05 ) , and the percentage of CD8 + cells was not significantly different from that of PBS control group ( 0 . 2P0.05 ) .



It can be seen from the above results that TB DNA vaccine mainly stimulates Th1 - type cell immunity and has no significant effect on inducing immune response of Th2 - type cells .
【學位授予單位】：武漢生物制品研究所
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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本文編號：2005584