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

Establishment and evaluation of the program of screening peptide mimotopes of blood group D carbohydrate antigen using phage display peptide library

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30min的洗脱液滴度；3轮清洗缓冲液中Tween20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8min时洗脱液滴度为（2．31±0．25）×10^6pfu／mL，洗脱4、12、16和30min时的洗脱液滴度与之相比明显下降（P〈0．01）。当洗脱时间为8min且3轮封闭缓冲液均为0．5％BSA时，3轮淘洗清洗缓冲液Tween20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Tween20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8min且3轮淘洗清洗缓冲液Tween20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8min，3轮淘洗的清洗缓）中液Tween20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 factors of screening peptide mimotopes of blood group D carbohydrate antigen using phage display peptide library, then to evaluate the best program for screening. Methods The phage titers of eluent in each experiment with different elution time （4, 8, 12, 16 and 30 minutes） were compared. The input/output ratio in each round panning with different concentration of Tween 20 in washing buffer were compared and the input/output ratio in each round panning with different blocking buffer types were also compared in order to analyze the effect of screening. The best program was used for screening to obtain the mimotopes of RhD blood type antigens. Afterwards, the antibody competition inhibition assay and DNA sequencing were conducted. Results The phage titers of eluent eluted after 8 minutes was （2. 31 ± 0. 25 ） × 10^6 pfu/mL, which was significantly different from the phage titers of eluent eluted after 4, 12, 16 and 30 minutes （P〈0. 01）. When the elution time was 8 minutes and the blocking buffer of 0. 5% BSA was used in each biopanning round, the input/output ratios were 1.69 × 10^7, 4. 95 × 10^5 and 9. 58 × 10^2 by using washing buffer of 0. 1% , 0. 5% and 0. 5% Tween 20. The results from using the washing buffer of 0. 1%, 0. 2%, 0. 5% Tween 20 were compared, the input/ output ratios were 2. 70 × 10^7, 4.57× 10^5, 1.14 × 10^3, respectively. The impact of concentration of Tween 20 was not obvious. The input/output ratios were 1.93 × 10^7, 6.44 × 10^3, 6. 23 × 10^1 by using blocking buffer of 0. 5% BSA, 1% Gelatin and 5% skimmed milk, when the elution time was 8 minutes and the washing buffer were 0. 1%, 0. 5% and 0. 5% Tween 20. The result was much better than the one with a blocking buffer of 0. 5% BSA in each biopanning round, which the input/ output ratios were 1.94 × 10^7, 1.66× 10^5 and 8.23× 10^2. By using the best program （the elution time is 8 minutes, the washing buffer of 0. 1%, 0. 5%, 0. 5% Tween 20 and the blocking buffer of 0. 5% BSA, 1% Gelatin and 5% skimmed milk in each biopanning round）, one major mimic peptides with about 50% inhibition ratio were obtained. The peptide sequence was ＂VHWDFRQWWQPS＂. Conclusion The best program of screening peptide mimotopes of blood group D carbohydrate antigen using phage display peptide library was ： eluted for 8 minutes, washing buffer of 0. 1%, 0. 5%, 0. 5% Tween 20 and blocking buffer of 0. 5% BSA, 1% Gelatin, 5% skimmed milk in each biopanning round. Satisfactory results are obtained.