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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(P<0.01)。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。
Objective To investigate the influencing factors of screening of D-peptide analogues by random phage diopeptide library to establish the optimal screening program. The titer of elution solution was compared with elution time of 4, 8, 12, 16 and 30 min respectively. Tween 20 concentrations of 0.1%, 0.2%, 0.5% and 0.1%, respectively, %, 0.5% BSA, 0.5% BSA, 1% gelatin, 5% skim milk per panning input / output ratio at 0.5%, and 3 rounds of blocking buffer, Output ratio to analyze the screening effect, draw the best screening program. The positive clones screened by the best screening scheme were tested for their DNA sequences and antibody competition inhibition test for the detection of D antigen mimotopes. Results The elution titer was (2.31 ± 0.25) × 10 ~ 6pfu / m L after 8 min of elution, and the titer of elution was significantly decreased at 4, 12, 16 and 30 min (P <0.01). When the elution time was 8 min and the three rounds of blocking buffer were both 0.5% BSA, the input / output ratios of three rounds of washing buffer Tween 20 were 0.1%, 0.5% and 0.5% 10 ~ 7, 4.95 × 10 ~ 5 and 9.58 × 10 ~ 2, respectively, and the input / output ratios were respectively 2.70 × 10 ~ 7 and 4.57 × 10 when the concentrations of Tween 20 and washing buffer were 0.1%, 0.2% and 0.5% ~ 5,1.14 × 10 ~ 3 compared to the screening effect has little effect. When the elution time was 8 min and the three rounds of wash buffer Tween 20 concentrations were 0.1%, 0.5% and 0.5% respectively, the three rounds of blocking buffer were 0.5% BSA, 1% gelatin and 5% skim milk respectively The input / output ratios were 1.93 × 10 ~ 7, 6.44 × 10 ~ 3 and 6.23 × 10 ~ 1, respectively, which were significantly better than the input / output ratios of 0.5% BSA in 3 rounds of washing buffer 1.94 × 10 ~ 7, 1.66 × 10 ~ 5, 8.23 × 10 ~ 2). A dodecapeptide sequence “VHWDFRQWWQPS ” with a suppression rate of about 50% was obtained through optimization. Conclusion The elution time, the composition of the washing buffer and the type of the blocking buffer have an impact on the screening efficiency. The elution time was 8 min. The three rounds of washing buffer Tween 20 at concentrations of 0.1%, 0.5%, 0.5% and three cycles of panning buffer were 0.5% BSA, 1% gelatin, 5% When the milk powder, the screening effect is better, can obtain more ideal blood group D antigen mimic polypeptide.