發(fā)布時(shí)間：2018-04-10 05:27

  本文選題：PTB-30m重組質(zhì)粒　切入點(diǎn)：轉(zhuǎn)化　出處：《重慶醫(yī)科大學(xué)》2006年碩士論文

【摘要】： 目的1.大量擴(kuò)增PTB-30m重組質(zhì)粒。2.評(píng)價(jià)DNA-85B和BCG序貫免疫能否誘導(dǎo)小鼠產(chǎn)生優(yōu)于常規(guī)BCG免疫的抗結(jié)核免疫保護(hù)作用。 方法1.將少量PTB-30m重組質(zhì)粒轉(zhuǎn)化入大腸桿菌DH5α中擴(kuò)增;用含氨芐青霉素的LB培養(yǎng)基進(jìn)行篩選;在含氨芐青霉素的LB液體培養(yǎng)基中擴(kuò)大培養(yǎng);用質(zhì)粒抽提試劑盒抽提得到高純度的PTB-30m重組質(zhì)粒(通過紫外分光光度計(jì)測(cè)A260或A260/A280);最后用HindⅢ和EcoreⅠ作雙酶切,1%瓊脂糖凝膠電泳(電壓170伏左右、時(shí)間約20分鐘)。2.用PTB-30m重組質(zhì)粒初次免疫C57BL/6小鼠,2周后用BCG加強(qiáng)免疫,分別于4周和8周后處死小鼠,制備脾淋巴細(xì)胞,用PPD刺激培養(yǎng)后分別用MTT法作淋巴細(xì)胞增殖轉(zhuǎn)化試驗(yàn);用ELISA試劑盒檢測(cè)脾淋巴細(xì)胞培養(yǎng)上清中IL-2和IFN-γ含量;用流式細(xì)胞儀檢測(cè)脾淋巴細(xì)胞表面CD25(IL-2Rα)的表達(dá)百分率。 結(jié)果1.共獲得高純度PTB-30m重組質(zhì)粒4856g,經(jīng)雙酶切后電泳有900bp左右片段存在,與Ag85B編碼基因相符合。2.實(shí)驗(yàn)組(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 偽 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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