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《Chinese Journal of Blood Transfusion》 2016-08
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Establishment and evaluation of the program of screening peptide mimotopes of blood group D carbohydrate antigen using phage display peptide library

WU Fan;ZHUANG Naibao;ZHANG Yinze;XU Hua;ZHOU Huayou;LI Kangsheng;Department of Microbiology and Immunology,Shantou University Medical College;Shenzhen Blood Center;Shaanxi Blood Center;Southern Medical University Nanfang Hospital;  
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 / m L,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% and0. 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% Tween20. 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 about50% 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.
【Fund】: 深圳市卫生系统科研项目(201401075)
【CateGory Index】: R457.11
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