本文選題：空腸彎曲菌 + 基因疫苗��； 參考：《遵義醫(yī)學院》2009年碩士論文

【摘要】： 目的:探討CJ pcDNA3.1(-)-peb1A免疫昆明種小鼠后的細胞免疫應答機制及對機體的保護性。方法:(1)重組質(zhì)粒pcDNA3.1(-)-peb1A的鑒定和大量制備。(2)重組質(zhì)粒pcDNA3.1(-)-peb1A免疫昆明種小鼠后,采集脾細胞和血清:①采用細胞ELISA法檢測小鼠脾T淋巴細胞膜表面分子CD3、CD4、CD8、CD25和CD28。②ELISA檢測小鼠血清中IL-2、IL-4、IL-10、IFN-γ和TGF-β的水平。③空腸彎曲菌攻擊實驗:實驗組分別于末次免疫后第26天用新鮮培養(yǎng)的CJ菌液灌胃免疫后小鼠,觀察灌胃后一周小鼠的發(fā)病及死亡情況,并做腸道分泌液細菌培養(yǎng)。結果:(1)重組質(zhì)粒pcDNA3.1(-)-peb1A PCR擴增產(chǎn)物經(jīng)瓊脂糖凝膠電泳,為單一條帶,大小為812bp;應用Qiagen plasmid Giga Kit大量抽提的純度為1.929,濃度為2064μg/ml。(2)T淋巴細胞膜表面分子水平的檢測結果顯示:①CD3中佐劑DNA組高于裸DNA組在第10天有顯著性差異(P＜0.05);②CD8中佐劑DNA組高于裸DNA組在第10、20天均有顯著性差異(P＜0.05),而裸DNA組高于兩對照組僅在第20天有顯著性差異(P＜0.05);③CD25中裸DNA組高于NS組在僅第10天有顯著性差異(P＜0.05),佐劑DNA組高于裸DNA組僅在第20天有顯著性差異(P＜0.05);④CD4及CD28中佐劑DNA組高于裸DNA組在第10、20天均有顯著性差異(P＜0.05);⑤在第10、20天各T淋巴細胞膜表面分子中佐劑DNA組高于兩對照組,有顯著性差異(P＜0.05)。(3)Th1類細胞因子IL-2、IFN-γ,在第10、20、30天,裸DNA組、佐劑DNA組、空載體組及NS組兩兩相比均無顯著性差異(P＞0.05);Th2類細胞因子IL-4、IL-10、TGF-β:①裸DNA組高于兩對照組,在IL-4、TGF-β的檢測中有顯著性差異(P＜0.05),在IL-10的檢測中裸DNA組高于NS組有顯著性差異(P＜0.05),其裸DNA組高于空載體組僅在第20天有顯著性差異(P＜0.05);②佐劑DNA組高于裸DNA組,在IL-4的檢測中有顯著性差異(P＜0.05),在IL-10的檢測中僅在第10、20天有顯著性差異(P＜0.05),在TGF-β的檢測中僅第20、30天有顯著性差異(P＜0.05);③佐劑DNA組高于兩對照組,有顯著性差異(P＜0.05)。(4)細菌攻擊實驗:由疾病指數(shù)可知,裸DNA組低于兩對照組,有顯著性差異(P＜0.05);佐劑DNA組低于裸DNA組,有顯著性差異(P＜0.05);由腸道分泌液細菌培養(yǎng)結果看出,裸DNA組的細菌指數(shù)低于兩對照組;佐劑DNA組低于裸DNA組。結論:(1)重組質(zhì)粒pcDNA3.1(-)-peb1A構建成功。(2)CJ pcDNA3.1(-)-peb1A疫苗能夠誘導有效的細胞免疫應答。
[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- 緯 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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

【參考文獻】

相關期刊論文 前10條

1 殷子惠;DNA疫苗的研究與應用[J];山東畜牧獸醫(yī);2003年02期

2 朱曉華;DNA疫苗免疫效果影響因素的分析[J];國外醫(yī)學(寄生蟲病分冊);2003年06期

3 廖國陽;基因免疫機理及安全性研究進展[J];國外醫(yī)學(免疫學分冊);2001年06期

4 秦衛(wèi)兵;Th1和Th2細胞在體內(nèi)的分化[J];國外醫(yī)學(免疫學分冊);2002年01期

5 姚新生;Th1與Th2研究概況及進展[J];國外醫(yī)學(免疫學分冊);2002年06期

6 劉文波,柴同杰,趙云玲;疫苗新秀─核酸疫苗[J];黑龍江畜牧獸醫(yī);2001年10期

7 張巍,朱榮生;基因免疫與基因疫苗[J];河南畜牧獸醫(yī);2002年07期

8 袁慧君,王三虎;基因免疫研究進展[J];河南職業(yè)技術師范學院學報;2004年03期

9 李成文;孫萬邦;余妍;李官成;宋明英;;空腸彎曲菌亞單位脂質(zhì)體佐劑疫苗誘導抗體產(chǎn)生規(guī)律的研究[J];瀘州醫(yī)學院學報;2005年06期

10 李成文;孫萬邦;余妍;馮勝軍;李官成;;空腸彎曲菌外膜蛋白亞單位脂質(zhì)體佐劑疫苗的免疫原性研究[J];免疫學雜志;2006年04期

，

本文編號：1963776