随机噬菌体肽库筛选血型D抗原模拟多肽方案的建立及初步验证

发布时间：2018-03-23 06:21

本文选题：噬菌体肽库　切入点：RhD抗原　出处：《中国输血杂志》2016年08期 　论文类型：期刊论文

【摘要】：目的探讨随机噬菌体十二肽库筛选血型D抗原模拟多肽的影响因素,以建立最佳筛选方案。方法对比洗脱时间分别为4、8、12、16和30 min的洗脱液滴度;3轮清洗缓冲液中Tween 20浓度分别为0.1%、0.2%、0.5%和分别为0.1%、0.5%、0.5%时每轮淘洗的投入/产出比,以及3轮封闭缓冲液均为0.5%BSA和分别为0.5%BSA、1%明胶、5%脱脂奶时每轮淘洗的投入/产出比,以分析筛选效果,得出最佳筛选方案。对采用最佳筛选方案筛选得到的阳性克隆测定其DNA序列和做抗体竞争抑制试验检测D抗原模拟表位。结果洗脱8 min时洗脱液滴度为(2.31±0.25)×10~6pfu/m L,洗脱4、12、16和30 min时的洗脱液滴度与之相比明显下降(P0.01)。当洗脱时间为8 min且3轮封闭缓冲液均为0.5%BSA时,3轮淘洗清洗缓冲液Tween 20浓度分别为0.1%、0.5%、0.5%时投入/产出比分别为1.69×10~7、4.95×10~5、9.58×10~2,与清洗缓冲液Tween 20浓度分别为0.1%、0.2%、0.5%时投入/产出比分别为2.70×10~7、4.57×10~5、1.14×10~3相比,对筛选效果影响不大。当洗脱时间为8 min且3轮淘洗清洗缓冲液Tween 20浓度分别为0.1%、0.5%、0.5%时,3轮封闭缓冲液分别为0.5%BSA、1%明胶、5%脱脂奶时投入/产出比分别为1.93×10~7、6.44×10~3、6.23×10~1,明显优于3轮淘洗封闭缓冲液均为0.5%BSA时的投入/产出比(分别为1.94×10~7、1.66×10~5、8.23×10~2)。通过优化方案筛选得到1个抑制率约为50%的十二肽序列"VHWDFRQWWQPS"。结论洗脱时间、清洗缓冲液成分以及封闭缓冲液类型对筛选效率有一定影响。洗脱时间为8 min,3轮淘洗的清洗缓冲液Tween 20浓度分别为0.1%、0.5%、0.5%和3轮淘洗的封闭缓冲液分别为0.5%BSA、1%明胶、5%脱脂奶粉时,筛选效果较优,可获得较理想的血型D抗原模拟多肽。
[Abstract]:Objective to investigate the factors influencing the screening of blood group D antigen mimic peptides by random phage dodecapeptide library. In order to establish the best screening scheme, the concentration of Tween 20 in the elution titer of 3 washing buffers with elution time of 4 ~ 8 ~ 12 ~ (12) and 30 min was 0. 1 ~ 0. 2% and 0. 5% and 0. 5%, respectively. The input-output ratio of each round of washing was compared with that of 0. 5%, 0. 5% and 0. 5%, respectively, when the elution time was 4 ~ 8 ~ 12 ~ 12 and 30 min, respectively. And the input-output ratio of each round of washing with 0.5%BSA and 0.5 BSA-1% gelatin and 5% skim milk, respectively, in order to analyze the screening effect. The best screening scheme was obtained. The DNA sequence of the positive clones screened by the best screening scheme was determined and the D-antigen mimic epitope was detected by the antibody competitive inhibition test. The elution titer was 2.31 卤0.25 脳 10~6pfu/m L after 8 min elution. When the elution time was 8 min and the three rounds of closed buffer were 0.5%BSA, the concentration of Tween 20 in washing buffer was 0.1% and 0.5%, respectively, and the input-output ratio was 1.69 脳 1074.95 脳 104.95 脳 109.58 脳 10 ~ 2, respectively, when the elution time was 8 min and the three rounds of closed buffer were all 0.5%BSA, the input-output ratio was 1.69 脳 104.95 脳 104.95 脳 104.95 脳 109.58 脳 10 ~ (2), respectively. The concentration of Tween _ (20) in washing buffer was 0.1 / 0.2, and the input-output ratio was 2.70 脳 10 ~ (7) / 4.57 脳 10 ~ (-5) ~ 1.14 脳 10 ~ (-3), respectively. When the elution time was 8 min and the concentration of Tween 20 in three washing buffers was 0.1 and 0.5, respectively, the input-output ratio was 1.93 脳 1036.44 脳 1036.23 脳 1036.23 脳 1036.23 脳 10 ~ (-1), respectively. The input-output ratio (1.94 脳 10 ~ (7)) 1.66 脳 10 ~ (5) ~ (5) ~ (8.23) 脳 10 ~ (2) ~ (2) of washing block buffer solution was obtained when 0.5%BSA was used. A dodecapeptide sequence "VHWDFRQWWQPS" with an inhibitory rate of about 50% was obtained by the optimized method. Conclusion the elution time is good. The washing buffer composition and the type of blocking buffer had certain influence on the screening efficiency. When the washing buffer Tween 20 concentration of washing buffer was 0.1% 0.5% and 0.5% and 0.5% respectively, the concentration of washing buffer 20 was 0.5% 0.5% and 0.5% Gelatin 5% skim milk powder, respectively, when the elution time was 8 mins and 3 rounds washing, respectively, the concentration of Tween 20 was 0.5% and 0.5%, respectively, when the washing buffer was 0.5BSA1% gelatin and 5% skim milk powder. The screening effect is better and the ideal peptide of blood group D antigen can be obtained.
【作者单位】：汕头大学医学院微生物学与免疫学教研室;深圳市血液中心;陕西省血液中心;南方医科大学南方医院;
【基金】：深圳市卫生系统科研项目(201401075)
【分类号】：R457.11

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