發(fā)布時(shí)間：2018-03-23 06:21

  本文選題：噬菌體肽庫(kù)　切入點(diǎn)：RhD抗原　出處：《中國(guó)輸血雜志》2016年08期 　論文類(lèi)型：期刊論文

【摘要】：目的探討隨機(jī)噬菌體十二肽庫(kù)篩選血型D抗原模擬多肽的影響因素,以建立最佳篩選方案。方法對(duì)比洗脫時(shí)間分別為4、8、12、16和30 min的洗脫液滴度;3輪清洗緩沖液中Tween 20濃度分別為0.1%、0.2%、0.5%和分別為0.1%、0.5%、0.5%時(shí)每輪淘洗的投入/產(chǎn)出比,以及3輪封閉緩沖液均為0.5%BSA和分別為0.5%BSA、1%明膠、5%脫脂奶時(shí)每輪淘洗的投入/產(chǎn)出比,以分析篩選效果,得出最佳篩選方案。對(duì)采用最佳篩選方案篩選得到的陽(yáng)性克隆測(cè)定其DNA序列和做抗體競(jìng)爭(zhēng)抑制試驗(yàn)檢測(cè)D抗原模擬表位。結(jié)果洗脫8 min時(shí)洗脫液滴度為(2.31±0.25)×10~6pfu/m L,洗脫4、12、16和30 min時(shí)的洗脫液滴度與之相比明顯下降(P0.01)。當(dāng)洗脫時(shí)間為8 min且3輪封閉緩沖液均為0.5%BSA時(shí),3輪淘洗清洗緩沖液Tween 20濃度分別為0.1%、0.5%、0.5%時(shí)投入/產(chǎn)出比分別為1.69×10~7、4.95×10~5、9.58×10~2,與清洗緩沖液Tween 20濃度分別為0.1%、0.2%、0.5%時(shí)投入/產(chǎn)出比分別為2.70×10~7、4.57×10~5、1.14×10~3相比,對(duì)篩選效果影響不大。當(dāng)洗脫時(shí)間為8 min且3輪淘洗清洗緩沖液Tween 20濃度分別為0.1%、0.5%、0.5%時(shí),3輪封閉緩沖液分別為0.5%BSA、1%明膠、5%脫脂奶時(shí)投入/產(chǎn)出比分別為1.93×10~7、6.44×10~3、6.23×10~1,明顯優(yōu)于3輪淘洗封閉緩沖液均為0.5%BSA時(shí)的投入/產(chǎn)出比(分別為1.94×10~7、1.66×10~5、8.23×10~2)。通過(guò)優(yōu)化方案篩選得到1個(gè)抑制率約為50%的十二肽序列"VHWDFRQWWQPS"。結(jié)論洗脫時(shí)間、清洗緩沖液成分以及封閉緩沖液類(lèi)型對(duì)篩選效率有一定影響。洗脫時(shí)間為8 min,3輪淘洗的清洗緩沖液Tween 20濃度分別為0.1%、0.5%、0.5%和3輪淘洗的封閉緩沖液分別為0.5%BSA、1%明膠、5%脫脂奶粉時(shí),篩選效果較優(yōu),可獲得較理想的血型D抗原模擬多肽。
[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 鹵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.
【作者單位】： 汕頭大學(xué)醫(yī)學(xué)院微生物學(xué)與免疫學(xué)教研室;深圳市血液中心;陜西省血液中心;南方醫(yī)科大學(xué)南方醫(yī)院;
【基金】：深圳市衛(wèi)生系統(tǒng)科研項(xiàng)目(201401075)
【分類(lèi)號(hào)】：R457.11

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