中试生产防龋DNA疫苗、结核DNA疫苗效力评价试验

发布时间：2018-06-11 14:24

本文选题：中试生产 + DNA疫苗　；参考：《武汉生物制品研究所》2010年硕士论文

【摘要】： DNA疫苗是近年来基因治疗研究中所衍生并发展起来的一个新的研究领域。它是指将编码某种蛋白质抗原的重组真核表达载体免疫机体,使外源基因在活体内表达,从而刺激免疫系统产生特异性的免疫应答。本论文以我所中试生产的防龋DNA疫苗和结核DNA疫苗为基础,对这两种疫苗免疫机体后诱导的免疫效能进行评价。实验一中试生产的防龋DNA疫苗效力评价试验本实验以武汉大学口腔医学院构建的靶向融合防龋DNA疫苗pGJA-P/VAX1为基础免疫定菌大鼠,研究靶向融合防龋DNA疫苗pGJA- P/VAX1的免疫防龋效果。将56只大鼠随机分为7组,接种变形链球菌并给予致龋饲料,按3因素(质粒,蛋白,佐剂)2水平(肌肉,粘膜免疫途径)析因设计方案免疫大鼠,分别于第0、2、4、6、8、10周采集大鼠血清和唾液样本。并建立大鼠血清IgG和唾液IgA的间接ELISA检测方法,对建立的方法进行方法学验证。完成了各组血清和唾液样本的检测。实验结束时处死大鼠,参照Keyes经典计分标准进行龋齿计分。SPSS软件分析结果表明: (1)疫苗免疫组特异性抗PAc血清IgG和唾液IgA抗体水平均显著高于空载体免疫组(P0.0 1),而龋齿记分显著低于空载体免疫组(P0.01 )。 (2)同一免疫途径中,DNA疫苗初免、蛋白加强组特异性抗PAc血清IgG水平显著高于单纯DNA疫苗免疫组(P0.01),特异性抗PAc唾液IgA抗体水平、龋齿计分无显著差异。 (3)同一免疫方案中,黏膜免疫组特异性抗PAc唾液IgA抗体水平高于肌肉免疫组,龋齿计分低于肌肉免疫组(P0.05),血清抗体水平无显著差异 (4)黏膜免疫中,佐剂蛋白加强组的血清IgG和唾液IgA最高,显著高于其他组(P0.01),龋齿计分单纯DNA不加佐剂组高于其他三组(P0.05)。由此得出结论:重组基因质粒pGJA-P能有效的抑制龋病的发生和发展。滴鼻法和肌肉注射法接种防龋DNA疫苗都可有效防龋,而滴鼻法较肌肉注射法能更有效的控制龋损的发生,降低龋坏程度。实验二中试生产的结核DNA疫苗初步效力试验以解放军309医院构建的编码Ag85A抗原的结核DNA疫苗为基础免疫小鼠,研究中试生产的TB DNA疫苗免疫效果。将4-6周龄的Balb/c小鼠20只随机分为2组,一组免疫TB DNA疫苗,另一组免疫PBS作为阴性对照,于免疫前采血,共免疫三次,在第三次免疫后3周摘眼球处死小鼠,收集全血,分离血清,进行血清特异性抗体检测和细胞因子(Interferon-γ、IL-4)检测;同时对每只小鼠取脾细胞进行特异性Interferon-γ分泌检测和脾细胞CD4+、CD8+百分率检测。结果统计分析显示: (1)TB DNA疫苗不能刺激小鼠机体产生显著的特异性抗体。 (2)疫苗免疫组血清细胞因子IFN-γ水平显著高于PBS对照组,而IL-4水平与PBS对照组之间无显著差异。 (3)酶联免疫斑点检测结果表明TB免疫组脾细胞特异性IFN-γ分泌水平高于PBS组(P0.05)。 (4)TB DNA疫苗免疫组CD4+细胞百分率高于PBS对照组有统计学差异(0.025P0.05); CD8+细胞百分率与PBS对照组没有统计学差异(0.2P0.25)。综合以上结果可以看出,TB DNA疫苗主要刺激机体Th1型细胞免疫,而对诱导机体产生Th2型细胞免疫应答没有显著的促进作用。
[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 - 纬 , IL - 4 ) were collected .

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

( 2 ) The level of IFN - 纬 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 - 纬 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 .
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】