空肠弯曲菌pcDNA3.1（-）-peb1A诱导的细胞免疫应答初步探讨

发布时间：2018-06-01 09:56

本文选题：空肠弯曲菌 + 基因疫苗　；参考：《遵义医学院》2009年硕士论文

【摘要】： 目的:探讨CJ pcDNA3.1(-)-peb1A免疫昆明种小鼠后的细胞免疫应答机制及对机体的保护性。方法:(1)重组质粒pcDNA3.1(-)-peb1A的鉴定和大量制备。(2)重组质粒pcDNA3.1(-)-peb1A免疫昆明种小鼠后,采集脾细胞和血清:①采用细胞ELISA法检测小鼠脾T淋巴细胞膜表面分子CD3、CD4、CD8、CD25和CD28。②ELISA检测小鼠血清中IL-2、IL-4、IL-10、IFN-γ和TGF-β的水平。③空肠弯曲菌攻击实验:实验组分别于末次免疫后第26天用新鲜培养的CJ菌液灌胃免疫后小鼠,观察灌胃后一周小鼠的发病及死亡情况,并做肠道分泌液细菌培养。结果:(1)重组质粒pcDNA3.1(-)-peb1A PCR扩增产物经琼脂糖凝胶电泳,为单一条带,大小为812bp;应用Qiagen plasmid Giga Kit大量抽提的纯度为1.929,浓度为2064μg/ml。(2)T淋巴细胞膜表面分子水平的检测结果显示:①CD3中佐剂DNA组高于裸DNA组在第10天有显著性差异(P＜0.05);②CD8中佐剂DNA组高于裸DNA组在第10、20天均有显著性差异(P＜0.05),而裸DNA组高于两对照组仅在第20天有显著性差异(P＜0.05);③CD25中裸DNA组高于NS组在仅第10天有显著性差异(P＜0.05),佐剂DNA组高于裸DNA组仅在第20天有显著性差异(P＜0.05);④CD4及CD28中佐剂DNA组高于裸DNA组在第10、20天均有显著性差异(P＜0.05);⑤在第10、20天各T淋巴细胞膜表面分子中佐剂DNA组高于两对照组,有显著性差异(P＜0.05)。(3)Th1类细胞因子IL-2、IFN-γ,在第10、20、30天,裸DNA组、佐剂DNA组、空载体组及NS组两两相比均无显著性差异(P＞0.05);Th2类细胞因子IL-4、IL-10、TGF-β:①裸DNA组高于两对照组,在IL-4、TGF-β的检测中有显著性差异(P＜0.05),在IL-10的检测中裸DNA组高于NS组有显著性差异(P＜0.05),其裸DNA组高于空载体组仅在第20天有显著性差异(P＜0.05);②佐剂DNA组高于裸DNA组,在IL-4的检测中有显著性差异(P＜0.05),在IL-10的检测中仅在第10、20天有显著性差异(P＜0.05),在TGF-β的检测中仅第20、30天有显著性差异(P＜0.05);③佐剂DNA组高于两对照组,有显著性差异(P＜0.05)。(4)细菌攻击实验:由疾病指数可知,裸DNA组低于两对照组,有显著性差异(P＜0.05);佐剂DNA组低于裸DNA组,有显著性差异(P＜0.05);由肠道分泌液细菌培养结果看出,裸DNA组的细菌指数低于两对照组;佐剂DNA组低于裸DNA组。结论:(1)重组质粒pcDNA3.1(-)-peb1A构建成功。(2)CJ pcDNA3.1(-)-peb1A疫苗能够诱导有效的细胞免疫应答。
[Abstract]:Objective: to investigate the mechanism of cellular immune response and its protective effect on Kunming mice immunized with CJ pcDNA3.1(-)-peb1A. Methods: 1) the identification of recombinant plasmid pcDNA3.1(-)-peb1A and the preparation of recombinant plasmid pcDNA3.1(-)-peb1A were used to immunize Kunming mice. Collection of spleen cells and serum from mice using ELISA assay for detection of CD3T lymphocyte surface molecule CD3C4CD8CD8CD25 and CD28.2ELISA for detection of IL-2mil IL-4mil IL-10IL-10IFN- 纬 and TGF- 尾 levels in mice. 3 attack test of Campylobacter jejuni: the experimental group was immunized at the last time. Mice were immunized with fresh cultured CJ bacteria on the 26th day after the epidemic. The incidence and death of mice were observed one week after gavage, and the bacteria were cultured in intestinal secretions. Results the amplified product of recombinant plasmid pcDNA3.1(-)-peb1A PCR was a single band by agarose gel electrophoresis. The purity of Qiagen plasmid Giga Kit was 1.929 and the concentration was 2064 渭 g/ml.(2)T. The results showed that the adjuvant in DNA group was higher than that in bare DNA group on the 10th day (P < 0.05). There was significant difference on the 10th day (P < 0.05) between the DNA group and the naked DNA group (P < 0.05), but there was significant difference on the 20th day only (P < 0.05) between the bare DNA group and the NS group (P < 0.05). The adjuvant DNA group was higher than the naked group (P < 0.05). There was significant difference only on the 20th day in the DNA group (P < 0.05) and in the adjuvant DNA group (P < 0.05) and the adjuvant DNA group was significantly higher than that in the bare DNA group on the 10th day (P < 0.05). On the 10th and 20th day, the adjuvant DNA group was higher than the two control groups. There was no significant difference (P > 0.05) between bare DNA group, adjuvant DNA group, empty carrier group and NS group (P > 0.05). IL-4, IL-10, TGF- 尾 1, TGF- 尾 1 in naked DNA group was higher than that in control group. There was a significant difference in the detection of IL-4 TGF- 尾 (P < 0.05). In the detection of IL-10, the naked DNA group was significantly higher than the NS group (P < 0.05), and the bare DNA group was higher than the empty carrier group only on the 20th day (P < 0.05), and the DNA group was higher than the bare DNA group (P < 0.05). There was significant difference in the detection of IL-4 (P < 0.05), in the detection of IL-10 (P < 0.05) only on the 10th day (P < 0.05), and in the detection of TGF- 尾 on day 2030 (P < 0.05). The difference was significant (P < 0.05) in the adjuvant DNA group, which was higher than that in the control group (P < 0.05). There was significant difference (P < 0.05) in bacterial attack test: according to the disease index, the bare DNA group was lower than the two control groups (P < 0.05), the adjuvant DNA group was lower than the bare DNA group (P < 0.05). The bacterial index of bare DNA group was lower than that of control group, and that of adjuvant DNA group was lower than that of bare DNA group. Conclusion the recombinant plasmid pcDNA3.1(-)-peb1A can induce an effective cellular immune response.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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