WO2020259673A1 - 一种利用噬菌体抗体库高效筛选纤维蛋白原的方法 - Google Patents

一种利用噬菌体抗体库高效筛选纤维蛋白原的方法 Download PDF

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WO2020259673A1
WO2020259673A1 PCT/CN2020/098424 CN2020098424W WO2020259673A1 WO 2020259673 A1 WO2020259673 A1 WO 2020259673A1 CN 2020098424 W CN2020098424 W CN 2020098424W WO 2020259673 A1 WO2020259673 A1 WO 2020259673A1
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phage
fibrinogen
culture plate
solution
screening
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杨明英
雷芳
帅亚俊
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Zhejiang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

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  • the invention relates to the field of blood coagulation materials, and specifically, is a method for efficiently screening fibrinogen using a phage antibody library.
  • Skin trauma is one of the most common injuries in life. It can be as small as daily abrasions, to large areas of trauma caused by surgery or even war, and bleeding needs to be stopped in time. Therefore, blood clotting function is very important for people.
  • the process of blood coagulation is essentially a process in which soluble fibrinogen in plasma becomes insoluble fibrin. When the external material comes into contact with the blood, the protein in the plasma will be adsorbed on the surface of the material. Prothrombin is activated through a cascade reaction to generate thrombin, which further catalyzes the conversion of fibrinogen into fibrin. Fibrin spontaneously forms a fibrous network and promotes wounds. Coagulation.
  • Fibrinogen or coagulation factor I
  • Fibrinogen is the most abundant coagulation factor in the blood, and it is also an acute reactive protein.
  • the enrichment of fibrinogen can facilitate the formation of fibrin network at the wound, and the phage that can target to bind human fibrinogen can specifically bind human fibrinogen to achieve fibrinogen in the blood. Enriched.
  • Phage display technology is to incubate the peptide library with the target protein for a period of time to wash away the unbound phage, eluting and amplifying the phage with strong binding ability, and then perform the next round of elution to obtain the binding to the target protein The process of the most capable phage.
  • the phage screened by this technology has good specificity and strong binding ability, and is suitable for screening phage targeting human fibrinogen.
  • the present invention invents a method for efficiently screening fibrinogen using a phage antibody library to obtain phage that can specifically bind to human fibrinogen.
  • the bacteriophage involved in the present invention is mainly used in the field of blood coagulation, and can specifically bind to human fibrinogen to promote blood coagulation.
  • the present invention discloses a method for efficiently screening fibrinogen using a phage antibody library, the steps adopted are as follows:
  • Coating protein Dilute human fibrinogen with a coating solution, take the human fibrinogen solution in a cell culture plate, place the plate in a humid box, and incubate overnight at a temperature of 2-8°C. Range guarantees protein vitality;
  • Bacteriophage binding aspirate the blocking solution of the culture plate in step 2), and wash 3-15 times repeatedly. Too few washing times, insufficient washing, and too many times will wash away strong binding phages, leading to subsequent steps Too few phages. Then add the liquid from the culture plate in step 3) to the washed culture plate, and shake it gently in a shaker for 1-4 hours. If the time is too short, the phage binding is not strong enough, and if the time is too long, non-specific binding will increase;
  • Phage amplification and counting Amplify and count the phage obtained in step 6), and dilute to 10 3 -10 13 pfu/mL. If the concentration is too low, there are too few phage screened to proceed, and the concentration is too high, phage enrichment The degree is not enough to get a better sequence, and the phage of step 7) can be obtained;
  • step 1) to step 7) two to five times. If the number of times is too few, the phage enrichment is not enough, the screening effect is not good, the number of times is too much, the cost and manpower increase, and it is not economical.
  • the phage dodecapeptide library in step 3) was changed to the phage obtained in step 7) of the previous round;
  • the first four groups of phage obtained in step 9) are further optimized to obtain the best binding phage, and the polypeptide displayed by the phage is the best binding polypeptide.
  • the protein coated in step 1) of the present invention is human fibrinogen
  • the phage thus obtained is a phage that specifically targets and binds to human fibrinogen.
  • the four groups of peptides displayed with the highest frequency of phage screened in the present invention are GPRPPSEHLQIT (SED ID NO: 1), LTPHKHHKHLHA (SED ID NO: 2), GPRPPLHLDSMR (SED ID NO: 3), HLHKHHLRSTTH ( SED ID NO: 4).
  • the optimal sequence of the phage screened in the present invention is GPRPPSEHLQIT (SED ID NO: 1).
  • the phage screened in the present invention can be added to the coagulation material to target and bind to human fibrinogen to enrich fibrinogen, thereby promoting the coagulation effect.
  • step 9) of the present invention the four phages obtained with the highest frequency were amplified, purified, and diluted to obtain four phage solutions of 10 3 -10 13 pfu/mL.
  • the phage display sequence with the highest absorbance is GPRPPSEHLQIT (SED ID NO:1), which proves that the phage is the best phage for targeted binding to human fibrinogen, and the polypeptide sequence GPRPPSEHLQIT is the best binding polypeptide.
  • the washing solution in step 4) and step 5) of the present invention is all PBST, and the elution solution in step 6) is 0.2M Glycine aqueous solution adjusted by HCl to pH 2.2, neutralized The solution is a 50 mM Tris aqueous solution adjusted to pH 9.1 by HCl.
  • the present invention has the following outstanding features:
  • the phage screened by the present invention can specifically bind to human fibrinogen and have strong binding power;
  • the phage screened in the present invention is easy to process and can be added to various coagulation materials to promote the enrichment of fibrinogen and enhance the coagulation effect by targeted binding to human fibrinogen;
  • the present invention has the characteristics of simple and easy-to-obtain reagents and high success rate.
  • Figure 1 is a diagram of the frequency of screening phage
  • Figure 2 is a diagram showing the results of ELISA optimization experiments
  • Figure 3 is a graph showing the results of coagulation index of phage added to silk fibroin gel.
  • Coating protein Dilute human fibrinogen with a coating solution, take the human fibrinogen solution in a cell culture plate, place the plate in a humid box, and incubate overnight at 4°C;
  • Bacteriophage binding aspirate the blocking solution of the culture plate in step 2), wash it repeatedly for 6 times, then add the liquid from the culture plate in step 3) to the washed culture plate, and shake it gently on a shaker for 2.5 hours;
  • Phage amplification and counting The phage obtained in step 6) is plated and counted, and Figure 1 is obtained. Amplify and count the phage obtained in step 6), and dilute to 10 10 pfu/mL to obtain the phage of step 7);
  • Coating protein Dilute human fibrinogen with a coating solution, take the human fibrinogen solution in a cell culture plate, place the plate in a humid box, and incubate overnight at 2°C;
  • Block the plate take out the plate in step 1) and add the blocking solution to incubate at 2°C;
  • Bacteriophage binding aspirate the blocking solution of the culture plate in step 2), wash it 3 times, then add the liquid from the culture plate in step 3) to the washed culture plate, and shake it gently on a shaker for 1 hour;
  • Phage amplification and counting the phage obtained in step 6) is amplified and counted, and diluted to 103 pfu/mL to obtain the phage of step 7);
  • step 1) to step 7) two more times.
  • the phage dodecapeptide library in step 3) is changed to the phage obtained in step 7) of the previous round;
  • Coating protein Dilute human fibrinogen with a coating solution, take the human fibrinogen solution in a cell culture plate, place the plate in a wet box, and incubate at 8°C overnight;
  • Bacteriophage binding aspirate the blocking solution of the culture plate in step 2), wash it 15 times, then add the liquid from the culture plate in step 3) to the washed culture plate, and shake it gently on a shaker for 4 hours;
  • Phage amplification and counting the phage obtained in step 6) is amplified and counted, and diluted to 10 13 pfu/mL to obtain the phage of step 7);
  • step 1) to step 7) five times.
  • the phage dodecapeptide library in step 3) is changed to the phage obtained in step 7) of the previous round;
  • Coating protein Dilute human fibrinogen with a coating solution, take the human fibrinogen solution in a cell culture plate, place the plate in a wet box, and incubate at 8°C overnight;
  • Bacteriophage binding aspirate the blocking solution of the culture plate in step 2), wash it repeatedly 15 times, then add the liquid from the culture plate in step 3) to the washed culture plate, and shake it gently on a shaker for 4 hours;
  • Phage amplification and counting the phage obtained in step 6) is amplified and counted, and diluted to 10 13 pfu/mL to obtain the phage of step 7);
  • step 1) to step 7) five times.
  • the phage dodecapeptide library in step 3) is changed to the phage obtained in step 7) of the previous round;
  • step 10) Mix the best bound phage obtained in step 9) into the coagulation material silk fibroin gel, and incubate it with fresh blood for 3 minutes to detect the hemoglobin content in the uncoagulated blood, which is the ratio of the total hemoglobin content before coagulation, that is, the coagulation index BCI, Figure 3 is obtained.
  • BCI index the ratio of the total hemoglobin content before coagulation

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Abstract

本发明公开了一种利用噬菌体抗体库高效筛选纤维蛋白原的方法，包括包被蛋白、封闭平板、噬菌体库去背景、噬菌体结合等步骤，本发明筛选出的噬菌体对比其余噬菌体，能够特异性结合人纤维蛋白原，且结合力强，易于加工，可以加入各种凝血材料中，通过靶向结合人纤维蛋白原，促使纤维蛋白原的富集，增强凝血效果，具有所用试剂简单易得，成功率高等特点。

Description

一种利用噬菌体抗体库高效筛选纤维蛋白原的方法 技术领域
本发明涉及凝血材料领域，具体地说，是一种利用噬菌体抗体库高效筛选纤维蛋白原的方法。
背景技术
皮肤创伤是生活中最常见的损伤之一，小到日常擦破皮，大到手术甚至战争中受到的大面积创伤导致出血，都需要及时止血，因而凝血功能对于人来说是非常重要的。凝血的过程本质上就是血浆中的可溶性纤维蛋白原变成不可溶的纤维蛋白的过程。当外界材料与血液接触之后，血浆中的蛋白会吸附在材料表面，凝血酶原通过级联反应被激活生成凝血酶，进一步催化纤维蛋白原转化为纤维蛋白，纤维蛋白自发形成纤维网，促进伤口凝血。纤维蛋白原即凝血因子Ⅰ，是血液中含量最高的凝血因子，也是一种急性反应蛋白。在凝血过程中，纤维蛋白原的富集能够有利于伤口处纤维蛋白网络的形成，而能够靶向结合人纤维蛋白原的噬菌体可以通过特异性结合人纤维蛋白原，实现血液中纤维蛋白原的富集。
噬菌体展示技术是通过将肽库与靶蛋白孵育一段时间，洗去未结合的噬菌体，将结合能力强的噬菌体洗脱并扩增后，再在进行下一轮的洗脱，得到与靶蛋白结合能力最强的噬菌体的过程。通过该技术筛选得到的噬菌体特异性好，结合能力强，适合用于筛选靶向人纤维蛋白原的噬菌体的筛选。
发明内容
本发明针对靶向人纤维蛋白原噬菌体领域的空缺，发明利用噬菌体抗体库高效筛选纤维蛋白原的方法，得到能特异性结合人纤维蛋白原的噬菌体。本发明涉及的噬菌体主要用于促凝血领域，能够特异性结合人纤维蛋白原，促进凝血。
本发明公开了一种利用噬菌体抗体库高效筛选纤维蛋白原的方法，所采用的步骤如下：
1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，在2-8℃温度下孵育过夜，该温度范围保证蛋白活力；
2)封闭平板：将步骤1)中的板取出，加入封闭液，在2-8℃温度下孵育；
3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤3-15次，洗涤次数太少，洗涤不够完全，次数过多，会洗掉结合力强的噬菌体，导致后续步骤的噬菌体过少。然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动1-4h，时间太短噬菌体结合不够牢固，时间太长非特异性结合会增多；
5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤3-15次，得到步骤5)的培养板；
6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置10-30min，时间太短洗脱不完全，时间太长噬菌体活力明显下降。然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
7)噬菌体扩增及计数：将步骤6)所得噬菌体进行扩增并计数，稀释至10 3-10 13pfu/mL，浓度太低，筛选得到噬菌体太少无法进行，浓度过高，噬菌体富集度不够，无法得到较好的序列，得到步骤7)的噬菌体；
8)再重复步骤1)至步骤7)二-五次，次数过少，噬菌体富集度不够，筛选效果不佳，次数过多，成本人力增加，不经济。在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，出现频数最高的四组噬菌体为筛选得到的前四组噬菌体；
10)对步骤9)所得到的的前四组噬菌体进行进一步优选，得到最佳结合噬菌体，该噬菌体展示的多肽为最佳结合多肽。
作为进一步地改进，本发明所述的步骤1)所包被的蛋白为人纤维蛋白原，由此获得的噬菌体为特异性靶向结合人纤维蛋白原的噬菌体。
作为进一步地改就，本发明筛选所得的噬菌体频数最高的四组展示的多肽为GPRPPSEHLQIT(SED ID NO：1)，LTPHKHHKHLHA(SED ID NO：2)，GPRPPLHLDSMR (SED ID NO：3)，HLHKHHLRSTTH(SED ID NO：4)。
作为进一步地改进，本发明筛选所得的噬菌体最优序列为GPRPPSEHLQIT(SED ID NO：1)。
作为进一步地改进，本发明筛选所得的噬菌体可以加入凝血材料中，靶向结合人纤维蛋白原，使纤维蛋白原富集，进而促进凝血效果。
作为进一步地改进，本发明所述的步骤9)中，对所得频数最高的四种噬菌体分别进行扩增纯化稀释，得到10 3-10 13pfu/mL的四种噬菌体溶液，对四种噬菌体进行ELISA实验，得到吸光度最高的噬菌体展示序列为GPRPPSEHLQIT(SED ID NO：1)，证明该噬菌体为靶向结合人纤维蛋白原的最佳噬菌体，多肽序列GPRPPSEHLQIT为最佳结合多肽。
作为进一步地改进，本发明所述的步骤4)步骤5)所述的洗涤液皆为PBST，步骤6)所述的洗脱液为0.2M Glycine水溶液由HCl调节PH至2.2而成，中和液为50mM Tris水溶液通过HCl调节PH为9.1而成。
与现有技术相比，本发明具有以下突出特点：
(1)本发明筛选出的噬菌体对比其余噬菌体，能够特异性结合人纤维蛋白原，且结合力强；
(2)本发明筛选所得噬菌体易于加工，可以加入各种凝血材料中，通过靶向结合人纤维蛋白原，促使纤维蛋白原的富集，增强凝血效果；
(3)本发明具有所用试剂简单易得，成功率高等特点。
附图说明
图1是筛选噬菌体频数图；
图2是ELISA优选实验结果图；
图3是噬菌体加入丝素凝胶的凝血指数结果图。
具体实施方式
下面通过具体实施例对本发明的技术方案作进一步的详细说明，以下实施例是对本发明的解释而本发明并不局限于以下实施例。
实施例1
1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，4℃孵育过夜；
2)封闭平板：将步骤1)中的板取出，加入封闭液4℃孵育；
3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤6次，然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动2.5h；
5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤10次，得到步骤5)的培养板；
6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置20min，然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
7)噬菌体扩增及计数：将步骤6)所得噬菌体进行涂板计数，得到图1。将步骤6)所得噬菌体进行扩增并计数，稀释至10 10pfu/mL，得到步骤7)的噬菌体；
8)再重复步骤1)至步骤7)三次，在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，得到图2。出现频数最高的四组噬菌体为筛选得到的前四组噬菌体，对它们进行进一步优选，得到最佳结合噬菌体，该噬菌体展示的多肽为最佳结合多肽。
实施例2
1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，2℃孵育过夜；
2)封闭平板：将步骤1)中的板取出，加入封闭液2℃孵育；
3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤3次，然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动1h；
5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤15次，得到步骤5) 的培养板；
6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置10min，然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
7)噬菌体扩增及计数：将步骤6)所得噬菌体进行扩增并计数，稀释至10 3pfu/mL，得到步骤7)的噬菌体；
8)再重复步骤1)至步骤7)二次，在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，出现频数最高的四组噬菌体为筛选得到的前四组噬菌体，他们所展示的多肽序列分别为GPRPPSEHLQIT(SED ID NO：1)，LTPHKHHKHLHA(SED ID NO：2)，GPRPPLHLDSMR(SED ID NO：3)，HLHKHHLRSTTH(SED ID NO：4)。对它们进行进一步优选，得到最佳结合噬菌体，该噬菌体展示的多肽为最佳结合多肽。
实施例3
1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，8℃孵育过夜；
2)封闭平板：将步骤1)中的板取出，加入封闭液8℃孵育；
3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤15次，然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动4h；
5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤3次，得到步骤5)的培养板；
6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置30min，然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
7)噬菌体扩增及计数：将步骤6)所得噬菌体进行扩增并计数，稀释至10 13pfu/mL，得到步骤7)的噬菌体；
8)再重复步骤1)至步骤7)五次，在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，出现频数最高的四组噬菌体为筛选得到的前四组噬菌体，对它们分别进行扩增纯化稀释，得到10 13pfu/mL的四种噬菌体溶液，对四种噬菌体进行ELISA实验，得到吸光度最高的噬菌体展示序列为GPRPPSEHLQIT(SED ID NO：1)。证明该噬菌体为靶向结合人纤维蛋白原的最佳噬菌体，多肽序列GPRPPSEHLQIT(SED ID NO：1)为最佳结合多肽。
实施例4
1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，8℃孵育过夜；
2)封闭平板：将步骤1)中的板取出，加入封闭液8℃孵育；
3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤15次，然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动4h；
5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤3次，得到步骤5)的培养板；
6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置30min，然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
7)噬菌体扩增及计数：将步骤6)所得噬菌体进行扩增并计数，稀释至10 13pfu/mL，得到步骤7)的噬菌体；
8)再重复步骤1)至步骤7)五次，在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，出现频数最高的四组噬菌体为筛选得到的前四组噬菌体，对它们分别进行扩增纯化稀释，得到10 3pfu/mL的四种噬菌体溶液，对四种噬菌体进行ELISA实验，得到吸光度最高的噬菌体展示序列为GPRPPSEHLQIT(SED ID NO：1)；
10)将步骤9)得到的最佳结合噬菌体混合进凝血材料丝素凝胶当中，与新鲜血液孵育3min，检测未凝血液中血红蛋白含量，占凝血前总血红蛋白含量的 比例，即凝血指数BCI，得到图3，BCI指数越低表示凝血效果越好。图示表明加入筛选所得最佳噬菌体的凝胶，比加入野生型噬菌体的凝胶以及单纯的凝胶，凝血效果更好。
最后，还需要注意的是，以上列举的仅是本发明的具体实施例子。显然，本发明不限于以上实施例子，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (7)

  1. 一种利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，所采用的步骤如下：
    1)包被蛋白：将人纤维蛋白原用包被液稀释，取该人纤维蛋白原溶液于细胞培养板中，将板置于湿盒中，在2-8℃温度下孵育过夜；
    2)封闭平板：将步骤1)中的板取出，加入封闭液，在2-8℃温度下孵育；
    3)噬菌体库去背景：将噬菌体十二肽库加入新的细胞培养板中，并在摇床平缓摇动；
    4)噬菌体结合：吸走步骤2)中培养板的封闭液，反复洗涤3-15次，然后将步骤3)中培养板的液体加入该洗涤后的培养板，在摇床平缓摇动1-4h；
    5)洗涤：将步骤4)中培养板的液体吸走，反复洗涤3-15次，得到步骤5)的培养板；
    6)洗脱：在步骤5)的培养板中加入洗脱液，室温静置10-30min，然后将液体转移至新的离心管中，加入中和液，得到噬菌体；
    7)噬菌体扩增及计数：将步骤6)所得噬菌体进行扩增并计数，稀释至10 3-10 13pfu/mL，得到步骤7)的噬菌体；
    8)再重复步骤1)至步骤7)二-五次，在进行重复的过程中，所述的步骤3)中噬菌体十二肽库改为上一轮步骤7)所得的噬菌体；
    9)对最后三轮步骤6)所得噬菌体进行测序，统计不同噬菌体出现的频数，出现频数最高的四组噬菌体为筛选得到的前四组噬菌体；
    10)对步骤9)所得到的前四组噬菌体进行进一步优选，得到最佳结合噬菌体，该噬菌体展示的多肽为最佳结合多肽。
  2. 根据权利要求1所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，步骤1)所包被的蛋白为人纤维蛋白原，由此获得的噬菌体为特异性靶向结合人纤维蛋白原的噬菌体。
  3. 根据权利要求1所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，筛选所得的噬菌体频数最高的四组展示的多肽为GPRPPSEHLQIT(SED ID NO：1)，LTPHKHHKHLHA(SED ID NO：2)，GPRPPLHLDSMR(SED ID NO：3)，HLHKHHLRSTTH(SED ID NO：4)。
  4. 根据权利要求1或3所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，筛选所得的噬菌体最优序列为GPRPPSEHLQIT(SED ID NO：1)。
  5. 根据权利要求1或2或3所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，筛选所得的噬菌体可以加入凝血材料中，靶向结合人纤维蛋白原，使纤维蛋白原富集，进而促进凝血效果。
  6. 根据权利要求1所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，所述的步骤9)中，对所得频数最高的四种噬菌体分别进行扩增纯化稀释，得到10 3-10 13pfu/mL的四种噬菌体溶液，对四种噬菌体进行ELISA实验，得到吸光度最高的噬菌体展示序列为GPRPPSEHLQIT(SED ID NO：1)。证明该噬菌体为靶向结合人纤维蛋白原的最佳噬菌体，多肽序列GPRPPSEHLQIT为最佳结合多肽。
  7. 根据权利要求1所述的利用噬菌体抗体库高效筛选纤维蛋白原的方法，其特征在于，步骤4)步骤5)所述的洗涤液皆为PBST，步骤6)所述的洗脱液为0.2M Glycine水溶液由HCl调节PH至2.2而成，中和液为50mM Tris水溶液通过HCl调节PH为9.1而成。
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