结核分枝杆菌T淋巴细胞结合短肽克隆的全细胞差减筛选

发布时间：2018-01-07 05:31

本文关键词：结核分枝杆菌T淋巴细胞结合短肽克隆的全细胞差减筛选　出处：《吉林农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究目的：以T淋巴细胞为靶标对结核分枝杆菌噬菌体随机肽库,进行3轮全细胞差减筛选,期望获得与T淋巴细胞特异性结合的短肽,为寻找能够激活T淋巴细胞的活性肽,进而为开发相应的结核病新型多肽疫苗提供一些实验依据。研究方法：利用尼龙毛柱法分离小鼠的T淋巴细胞,以结核分枝杆菌H37Rv免疫Balb/c小鼠的T淋巴细胞为靶细胞,以生理盐水注射的Balb/c小鼠的T淋巴细胞为阴性吸附细胞,对结核分枝杆菌噬菌体随机肽库进行3轮全细胞差减筛选,在第一、二、三轮筛选中,结核分枝杆菌噬菌体随机肽库与免疫组T淋巴细胞的孵育时间分别为2h、1.5h、1h；与正常细胞孵育时间分别为2h、2.5h、3h；洗脱液的浓度分别为1%、3%、5%。蓝白斑筛选阳性克隆,提取纯化阳性克隆的单链DNA进行电泳鉴定,并观察阳性克隆对小鼠淋巴细胞转化的影响。研究结果：结核分枝杆菌H37Rv免疫的Balb/c小鼠血清抗PPD IgG水平在第六周时达到1：3200；第2周、第4周、第6周的对照组和免疫组之间差异显著(p0.05)；除笫2周差异不极显著外、第4周、第6周的对照组和免疫组之间差异极显著(p0.01),抗体水平的增加说明已成功获得免疫小鼠。尼龙毛柱分离T淋巴细胞的回收率为30%,活细胞比率为93%,得到了高活力的T淋巴细胞。3轮全细胞差减筛选后噬菌体得到了富集,噬菌体的回收率从4.0×10-55增加到52,获得的阳性克隆滴度为1.3×104/ml。蓝白斑筛选得到了阳性克隆,阳性克隆单链DNA电泳条带一致。阳性克隆对免疫组T淋巴细胞转化的影响明显大于其他刺激物,其刺激指数最大为3.5。结论：成功利用尼龙毛柱分离到T淋巴细胞,其回收率为30%,活细胞比率为93%；3轮全细胞差减筛选后获得了阳性克隆,阳性克隆滴度为1.3×104/ml；阳性克隆能够有效刺激T淋巴细胞活化。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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