一种血红蛋白提取液的制备方法技术领域 本发明属于血红蛋白的分离纯化工艺技术领域，具体地说，涉及血红蛋白分离纯化工艺中制备血红蛋白提取液的方法。 Technical Field method for preparing a hemoglobin extract  Separation and Purification The present invention belongs to the hemoglobin, specifically, the method extracts prepared hemoglobin hemoglobin separation and purification processes involved. 背景技术 血红蛋白的分离纯化工艺是携氧材料研制的重要工艺步骤。  The separation and purification of hemoglobin is an important process steps carrying oxygen material developed. 已有的以无基质血红蛋白为载体的携氧材料中，除基因工程重组表达的血红蛋白外，大部分血红蛋白都来源于动物，包括牛、猪、人等，要利用这些动物血红蛋白，首先需要一种低成本、易标准化的血红蛋白纯化方法。 Existing in stroma-free hemoglobin oxygen carrying support material, in addition to the expression of recombinant hemoglobin, most of the hemoglobin is derived from animals, including cattle, pigs, humans, etc., to make use of these animals hemoglobin, a need for a first low cost, easy to hemoglobin purification process standardization.  现有的血红蛋白纯化方法主要有加热法、超滤法、萃取法、阴离子交换柱层析法等。  The conventional methods are heating hemoglobin purification, ultrafiltration, extraction, anion exchange column chromatography and the like. 加热法和萃取法对血红蛋白的空间结构产生的影响不可避免，且萃取法引入的有机溶剂也是一种潜在的危险因素；超滤法虽然便于进行密闭的流水线生产，但超滤膜需要经常更换，成本高昂；阴离子交换柱层析法成本低，对血红蛋白的空间结构没有影响，且不引入有机溶剂，因而是最有前景的血红蛋白纯化方法。 Effect of Heat and extraction of spatial structure of hemoglobin produced is inevitable, and the organic solvent extraction method is introduced as a potential risk factor; ultrafiltration though facilitate closed production lines, but ultrafiltration membranes require frequent replacement, high cost; low anion exchange column chromatography costs, no effect on the spatial structure of hemoglobin, without the introduction of an organic solvent, the hemoglobin purification process is therefore the most promising.  阴离子交换柱层析法对用于上柱的蛋白提取液有以下两个方面的要求：（1)蛋白提取液必须不含大颗粒物，一般要求经O. 45ΜΠ1滤膜过滤；（2)蛋白提取液中的蛋白成分应尽可能少，并尽可能减少与阴离子交换柱层析介质作用强烈的成分的含量。  The anion exchange column chromatography on protein extracts on the column for the following two requirements: (1) protein extracts must be free of large particulate matter, generally require by O. 45ΜΠ1 membrane filtration; (2 ) protein extract the protein components should be as small as possible and to minimize the content of the strong role of the media component column chromatography and anion exchange.  传统的用于阴离子交换层析的血红蛋白提取液的制备方法，一般包括以下步骤： (I)将压积红细胞与低渗溶液(通常是水）按照一定体积比混合以制备低渗环境， 静置过夜使红细胞破碎； (2)离心红细胞破碎液，将上清转移到新的离心管； (3)将上清以O. 45ΜΠ1滤膜过滤，得到血红蛋白提取液； (4)将血红蛋白提取液置换至阴离子交换层析平衡缓冲液体系。 Preparation Method  The conventional anion exchange chromatography is used to extract hemoglobin, generally comprises the steps of:  (I) the hematocrit of red blood cells with a hypotonic solution (usually water) in accordance with a volume mixing ratio to prepare hypotonic environment, allowed to stand overnight to allow the red blood cells break;  (2) erythrocyte lysate was centrifuged, the supernatant was transferred to a new centrifuge tube;  (3) The supernatant O. 45ΜΠ1 membrane filtration to give Hemoglobin extract;  (4) The hemoglobin replacement extract to an anion exchange chromatography equilibration buffer system.  通过上述传统方法制备血红蛋白提取液，除了红细胞破碎液体积大，不利于后续步骤开展以外，更重要的是存在以下两个方面的问题： (I)得到的血红蛋白提取液难于通过O. 45ΜΠ1滤膜，因此需要频繁地更换滤膜，更换滤膜的过程提高了提取液中高铁血红蛋白的含量； (2)得到的血红蛋白提取液中含有较多的磷脂及脂蛋白，以填料能耐受的最大强度的洗脱溶液也不能将其洗脱，必须执行清洁程序。  prepared by the above conventional method hemoglobin extract, in addition to the large volume of red blood cell lysates, is not conducive to the subsequent step carried out outside, more important is the problem of the following two aspects: hemoglobin extract  (I) obtained is difficult by O. 45ΜΠ1 membrane, and therefore require frequent replacement of membranes, membrane replacement process increases the extract content of methemoglobin;  (2) hemoglobin extract obtained contains more phospholipids and lipoproteins to elute the maximum intensity of the filler solution can tolerate nor can it afford cleaning procedure must be performed. 清洁程序需要耗费大量的乙醇或异丙醇，且时间较长，不适宜大规模生产。 Cleaning procedure takes a lot of ethanol or isopropanol, and for a long time, not suitable for mass production. 发明内容 本发明的目的是提供一种适用于阴离子交换柱层析法大量生产要求的血红蛋白提取液的制备方法，采用该方法制备的血红蛋白提取液易于通过O. 45ΜΠ1滤膜，脂蛋白及磷脂含量少。 SUMMARY OF THE INVENTION  The object of the present invention is to provide a method suitable for preparing the anion column chromatography mass production requirements hemoglobin switching extract, the extract using hemoglobin readily prepared by O. 45ΜΠ1 membrane, lipoprotein and less phospholipid content.  为实现上述目的，本发明所述血红蛋白提取液的制备方法，包括以下步骤，各步骤均在O〜10°C的环境温度条件下进行： (I)将压积红细胞置于透析袋中，用缓冲液经低渗透压破碎，得到红细胞破碎液； (2)将与0°C时终饱和度为25%对应量的硫酸铵加入红细胞破碎液中，待硫酸铵充分溶解后以4000 G离心； (3)向步骤（2)离心所得的上清液中加入与0°C时终饱和度为60%对应量的硫酸铵，待硫酸铵充分溶解后以6000G离心； (4)将步骤（3)离心所得的蛋白沉淀重悬后转入透析袋中，用缓冲液透析去除硫酸铵； (5)经过透析的蛋白液用缓冲液按体积比为1:5进行稀释，然后以10000G离心，去除颗粒物。  To achieve the above object, the present invention is a method for preparing the hemoglobin extraction liquid, comprising the following steps, the steps were carried out under ambient temperature conditions O~10 ° C were:  (I) the red blood cell hematocrit set dialysis bag by hypotonic buffer crushed to obtain red blood cell lysate;  (2) at 0 ° C with a final saturation of 25% corresponds to an amount of ammonium sulfate added erythrocyte lysate, and to be sulfuric acid After ammonium fully dissolved in 4000 G centrifuge;  (3) to step (2) a final saturation when added to the resulting supernatant was centrifuged at 0 ° C with 60% corresponding to the amount of ammonium sulfate, ammonium sulfate until fully dissolved After centrifugation to 6000G;  (4) The step (3) the resulting protein precipitate after centrifugation was resuspended into the dialysis bags, dialyzed to remove ammonium sulfate buffer;  (5) through the dialyzed protein solution using buffer volume ratio of 1: 5 diluted, and then centrifuged at 10000G, to remove particulate matter.  本发明的步骤（I)中采用透析的方式制造低渗透压环境使红细胞破碎，有效减小了红细胞破碎液的体积，增加了溶液的密度，从而为后续的硫酸铵分级分离创造了条件；并且因为红细胞破碎液体积小，极大的节约了硫酸铵的用量。  Step (I) of the present invention is manufactured using dialysis hypotonic environment in which red blood cells break, effectively reducing the volume of red blood cell lysates, increasing the density of the solution, thus creating for subsequent ammonium sulfate fractionation conditions; and because the volume of red blood cell lysates small, a great saving of the amount of ammonium sulfate. 采用步骤（2)和步骤（3)的两步硫酸铵沉淀操作，制备不同的溶液密度，使得脂蛋白和磷脂在相应密度与离心力条件下与血红蛋白处于离心管中的不同位置，因而使绝大部分的脂蛋白以及部分杂蛋白得以去除。 Using step (2) and step (3) two-step operation ammonium sulfate precipitation, prepared different solution density, making lipoproteins and phospholipids in the corresponding conditions and hemoglobin density centrifugal force in a centrifuge tube at different positions, thus the vast lipoprotein and some miscellaneous parts of the protein to be removed.  实践表明，经过本发明方法制备的血红蛋白提取液，与传统的用于阴离子交换层析的血红蛋白提取液制备方法相比，其脂蛋白与憐脂含量残留率小于2%,易于通过O. 45Mm 滤膜；且硫酸铵用量少，纯化成本低，血红蛋白回收率较高，具有很好的应用前景。  The practice shows that hemoglobin extract prepared through the method of the present invention, hemoglobin extract preparation method and the traditional anion exchange chromatography is used compared to the lipoprotein and pity residual fat content less than 2%, easy by O . 45Mm membrane; and ammonium sulfate with less, low cost purification, high hemoglobin recovery, has good prospects.  附图说明 附图是本发明与传统的用于阴离子交换层析的血红蛋白提取液制备方法相比，制备的血红蛋白提取液的SDS-PAGE分析结果对照图； 图中：M表示蛋白分子量标准； C表示传统方法制备的血红蛋白提取液； D表示本发明方法制备的血红蛋白提取液。  BRIEF DESCRIPTION OF DRAWINGS  FIG extract hemoglobin production method of the present invention is used with conventional anion exchange chromatography compared to extracts prepared from hemoglobin by SDS-PAGE analysis with reference to Figs;  FIG. where: M represents protein molecular weight standards;  C represents hemoglobin extract prepared by conventional methods;  D represents hemoglobin extract prepared by the method of the present invention. 具体实施方式 下面结合附图和实施例对本发明作进一步说明。 DETAILED DESCRIPTION  below in conjunction with the accompanying drawings and the embodiment of the present invention will be further described below.  在O〜10 V的环境温度条件下依次进行以下操作： (I)将压积红细胞在平均截留分子量为60 kDa的透析袋中经低渗透压破碎得到红细胞破碎液，透析所用缓冲液为经过脱氧处理的Tris-HCl缓冲液，缓冲液的Tris base浓度为100 mM，pH值为8. O ； (2)将与0°C时终饱和度为25%对应量的硫酸铵加入红细胞破碎液中，待硫酸铵充分溶解后以4000 G离心； (3)向步骤⑵中离心所得的上清液中加入与0°C时终饱和度为60%对应量的硫酸铵，待硫酸铵充分溶解后以6000G离心； (4)将步骤（3)所得的沉淀蛋白重悬后转入平均截留分子量为60 kDa的透析袋中透析以除去硫酸铵，透析所用缓冲液为经过脱氧处理的Tris-HCl，NaCl缓冲液，缓冲液的Tris base浓度为55 mM, NaCl浓度为30 mM,缓冲液的pH值为8. 5 ； (5)经过透析的蛋白液用缓冲液按体积比为1:5的比例进行稀释，然后以10000离心去除颗粒物，所用缓冲液是经过脱氧处理的Tris-HCl，NaCl缓冲液，缓冲液的Tris base浓度为55 mM, NaCl浓度为30 mM,缓冲液的pH值为8. 5。  carried out at ambient temperature conditions O~10 V followed by the following:  (I) the hematocrit of red blood cells in the average molecular weight of 60 kDa cut-off dialysis bags get crushed by hypotonic red blood cell lysates, dialysis the concentration of Tris base buffer was used after deoxidation processing Tris-HCl buffer, the buffer is 100 mM, pH value of 8. O;  (2) with the 0 ° C to a final saturation of 25% corresponds to the amount of ammonium sulfate was added 4000 G erythrocyte lysate was centrifuged, and to be fully dissolved in ammonium sulfate; when  (3) To the supernatant obtained in step ⑵ centrifuged 0 ° C was added with a final saturation of 60% After the corresponding amount of ammonium sulfate, ammonium sulfate until fully dissolved to 6000G centrifugation;  (4) Step (3) The resulting protein precipitate was resuspended into an average molecular weight of 60 kDa cutoff dialysis bag dialyzed to remove sulfate ammonium, dialysis buffer was used after deoxidation processing Tris-HCl, NaCl buffer, Tris base buffer concentration was 55 mM, NaCl at a concentration of 30 mM, pH of the buffer is 8.5;  (5 ) dialyzed protein was buffer volume ratio of 1: 5 diluted and then 10,000 centrifuged to remove particulates, the buffer is through deoxidation of Tris-HCl, NaCl buffer, buffer Tris base concentration to 55 mM, NaCl at a concentration of 30 mM, pH of the buffer is 8.5.  本实施例，利用本发明方法制备血红蛋白提取液，血红蛋白回收率为87%，脂蛋白及磷脂残留量为I. 5%，易于通过O. 45Mffl滤膜，非常适合于阴离子交换柱层析法大量生产的要求。  In this example, using the method of the present invention are prepared extract hemoglobin, hemoglobin recovery rate was 87%, lipoproteins and phospholipids remaining amount is I. 5%, by O. 45Mffl easy to filter and is suitable for anion exchange column chromatography mass production requirements.  说明书附图是本发明方法与传统方法制备的血红蛋白提取液的SDS-PAGE对照分析图。  The accompanying drawings is a SDS-PAGE analysis of the control method of the present invention and the conventional FIG prepared extract hemoglobin. 从图中可以看出，采用本发明方法制备的血红蛋白提取液中蛋白成分得到有效的减少，且血红蛋白含量提高。 As can be seen from the figure, using hemoglobin extract prepared by the method of the protein component can be effectively reduced, and the hemoglobin content increased.