本文關(guān)鍵詞：結(jié)核分枝桿菌T淋巴細(xì)胞結(jié)合短肽克隆的全細(xì)胞差減篩選　出處：《吉林農(nóng)業(yè)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 結(jié)核分枝桿菌噬菌體隨機(jī)肽庫 全細(xì)胞差減篩選 T淋巴細(xì)胞 短肽

【摘要】：研究目的：以T淋巴細(xì)胞為靶標(biāo)對結(jié)核分枝桿菌噬菌體隨機(jī)肽庫,進(jìn)行3輪全細(xì)胞差減篩選,期望獲得與T淋巴細(xì)胞特異性結(jié)合的短肽,為尋找能夠激活T淋巴細(xì)胞的活性肽,進(jìn)而為開發(fā)相應(yīng)的結(jié)核病新型多肽疫苗提供一些實(shí)驗(yàn)依據(jù)。 研究方法：利用尼龍毛柱法分離小鼠的T淋巴細(xì)胞,以結(jié)核分枝桿菌H37Rv免疫Balb/c小鼠的T淋巴細(xì)胞為靶細(xì)胞,以生理鹽水注射的Balb/c小鼠的T淋巴細(xì)胞為陰性吸附細(xì)胞,對結(jié)核分枝桿菌噬菌體隨機(jī)肽庫進(jìn)行3輪全細(xì)胞差減篩選,在第一、二、三輪篩選中,結(jié)核分枝桿菌噬菌體隨機(jī)肽庫與免疫組T淋巴細(xì)胞的孵育時間分別為2h、1.5h、1h；與正常細(xì)胞孵育時間分別為2h、2.5h、3h；洗脫液的濃度分別為1%、3%、5%。藍(lán)白斑篩選陽性克隆,提取純化陽性克隆的單鏈DNA進(jìn)行電泳鑒定,并觀察陽性克隆對小鼠淋巴細(xì)胞轉(zhuǎn)化的影響。 研究結(jié)果：結(jié)核分枝桿菌H37Rv免疫的Balb/c小鼠血清抗PPD IgG水平在第六周時達(dá)到1：3200；第2周、第4周、第6周的對照組和免疫組之間差異顯著(p0.05)；除笫2周差異不極顯著外、第4周、第6周的對照組和免疫組之間差異極顯著(p0.01),抗體水平的增加說明已成功獲得免疫小鼠。尼龍毛柱分離T淋巴細(xì)胞的回收率為30%,活細(xì)胞比率為93%,得到了高活力的T淋巴細(xì)胞。3輪全細(xì)胞差減篩選后噬菌體得到了富集,噬菌體的回收率從4.0×10-55增加到52,獲得的陽性克隆滴度為1.3×104/ml。藍(lán)白斑篩選得到了陽性克隆,陽性克隆單鏈DNA電泳條帶一致。陽性克隆對免疫組T淋巴細(xì)胞轉(zhuǎn)化的影響明顯大于其他刺激物,其刺激指數(shù)最大為3.5。 結(jié)論：成功利用尼龍毛柱分離到T淋巴細(xì)胞,其回收率為30%,活細(xì)胞比率為93%；3輪全細(xì)胞差減篩選后獲得了陽性克隆,陽性克隆滴度為1.3×104/ml；陽性克隆能夠有效刺激T淋巴細(xì)胞活化。
[Abstract]:Objective: to study three rounds of whole-cell subtractive screening of Mycobacterium tuberculosis phage random peptide library targeting T lymphocytes in order to obtain a short peptide specifically bound to T lymphocytes. In order to search for the active peptides that can activate T lymphocytes, and then provide some experimental basis for the development of the corresponding new peptide vaccine for tuberculosis. Methods: the T lymphocytes of mice were isolated by nylon hair column method. The T lymphocytes of Balb/c mice immunized with Mycobacterium tuberculosis H37Rv were used as target cells. Using T lymphocytes of Balb/c mice injected with normal saline as negative adsorption cells, three rounds of whole-cell subtractive screening were carried out on the phage random peptide library of Mycobacterium tuberculosis, and the first, second and third rounds of screening were carried out. The incubation time of Mycobacterium tuberculosis phage random peptide library with T lymphocytes in the immunized group was 2 h, 1.5 h and 1 h, respectively. The incubation time with normal cells was 2 h, 2.5 h and 3 h, respectively. The concentration of eluant was 1 ~ 3 and 5% respectively. The positive clones were screened by blue and white spot, the single strand DNA of purified positive clones were extracted and identified by electrophoresis, and the effect of positive clones on lymphocyte transformation in mice was observed. Results: the serum anti-#en1# IgG level of Balb/c mice immunized with Mycobacterium tuberculosis H37Rv reached 1: 3200 at 6th weeks. At week 2, week 4 and week 6, the difference between the control group and the immunized group was significant (p 0.05). Except for the second week, the difference between the control group and the immune group was significant at the 4th week and the 6th week (p0.01). The increase of antibody level showed that the immunized mice were successfully immunized. The recovery rate of T lymphocytes isolated by nylon column was 30 and the living cell ratio was 93%. Three rounds of whole-cell subtractive screening of T lymphocytes with high activity were obtained and the phage was enriched. The recovery rate of phage increased from 4.0 脳 10 ~ (-55) to 52. The titer of positive clones was 1.3 脳 10 4 / ml. The positive clones were obtained by blue and white spot screening. The single strand DNA electrophoresis bands of positive clones were consistent, and the effect of positive clones on T lymphocyte transformation in immunized group was significantly greater than that of other stimuli, and the maximum stimulating index was 3.5. Conclusion: the T lymphocytes were successfully isolated with nylon capillary column. The recovery rate was 30% and the living cell ratio was 93%. After three rounds of whole cell subtractive screening, positive clones were obtained, the titer of positive clones was 1.3 脳 104 / ml; Positive clones can effectively stimulate the activation of T lymphocytes.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

【引證文獻(xiàn)】

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本文編號：1391185