DNA-85B增强BCG免疫小鼠抗结核免疫保护作用的探讨

发布时间：2018-04-10 05:27

  本文选题：PTB-30m重组质粒　切入点：转化　出处：《重庆医科大学》2006年硕士论文

【摘要】： 目的1.大量扩增PTB-30m重组质粒。2.评价DNA-85B和BCG序贯免疫能否诱导小鼠产生优于常规BCG免疫的抗结核免疫保护作用。 方法1.将少量PTB-30m重组质粒转化入大肠杆菌DH5α中扩增;用含氨苄青霉素的LB培养基进行筛选;在含氨苄青霉素的LB液体培养基中扩大培养;用质粒抽提试剂盒抽提得到高纯度的PTB-30m重组质粒(通过紫外分光光度计测A260或A260/A280);最后用HindⅢ和EcoreⅠ作双酶切,1%琼脂糖凝胶电泳(电压170伏左右、时间约20分钟)。2.用PTB-30m重组质粒初次免疫C57BL/6小鼠,2周后用BCG加强免疫,分别于4周和8周后处死小鼠,制备脾淋巴细胞,用PPD刺激培养后分别用MTT法作淋巴细胞增殖转化试验;用ELISA试剂盒检测脾淋巴细胞培养上清中IL-2和IFN-γ含量;用流式细胞仪检测脾淋巴细胞表面CD25(IL-2Rα)的表达百分率。 结果1.共获得高纯度PTB-30m重组质粒4856g,经双酶切后电泳有900bp左右片段存在,与Ag85B编码基因相符合。2.实验组(D-B
[Abstract]:Objective 1.A large number of PTB-30m recombinant plasmids.To evaluate whether DNA-85B and BCG Sequential immunization can induce mice to produce antituberculous immune protective effect which is superior to routine BCG immunization.Method 1.A small amount of PTB-30m recombinant plasmid was transformed into Escherichia coli DH5 伪 for amplification, and was screened on LB medium containing ampicillin, and was expanded in LB liquid medium containing ampicillin.High purity PTB-30m recombinant plasmids were obtained by using plasmid extraction kit (A260 or A260 / A280) detected by UV spectrophotometer, and 1% agarose gel electrophoresis with double enzyme digestion with Hind 鈪，

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