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 The extract one kind of hemoglobin  Technical Field separation and purification of hemoglobin present invention pertains, in particular, to a method of separation and purification process of preparing the hemoglobin hemoglobin extract. 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。  hemoglobin separation and purification process is an important step in the development of an oxygen-carrying material. 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。 Conventional oxygen-carrying hemoglobin is stroma-free material carrier, in addition to the expression of Recombinant hemoglobin, hemoglobin are derived from most animals, including cattle, pigs, humans, etc., to the use of these animals hemoglobin, a need for a first low cost, easy normalized hemoglobin purification process.  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。  Hemoglobin conventional purification methods are heating, 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 heating and extraction of the spatial structure of hemoglobin produced inevitably, and the introduced organic solvent extraction method is also a potential risk factor; ultrafiltration closed while facilitating the production line, but require frequent replacement membrane, high cost; low cost anion exchange chromatography column, no effect on the spatial structure of hemoglobin, without the introduction of an organic solvent, which is the most promising method for hemoglobin purification.  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 has the following two requirements of a protein extract on the column: (1) protein extract be free of large particles, typically O. 45ΜΠ1 required by membrane filtration; (2 ) protein component of the protein extracts should be as small as possible, and to reduce the content of component chromatographic medium strong anion exchange column and the action as possible.  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。 The method of preparing hemoglobin extract  conventional for anion exchange chromatography, typically comprising the steps of:  (I) the packed red cells with a hypotonic solution (usually water) was prepared according to a volume ratio of hypotonic environment, allowed to stand overnight broken erythrocytes;  (2) erythrocyte homogenate was centrifuged, the supernatant was transferred to a new centrifuge tube;  (3) the supernatant O. 45ΜΠ1 membrane filtration, to give hemoglobin extract;  (4) substituted hemoglobin 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。  By the above-described conventional method of preparing hemoglobin extract, in addition to large volume of red blood cell homogenate, is not conducive to the subsequent step carried out except that the more important issue is the presence of the following two aspects: hemoglobin extract  (I) obtained is difficult by O. 45ΜΠ1 membrane, it is necessary to frequently replace the filter, the filter replacement process increases the extract content of methemoglobin;  (2) hemoglobin extract obtained contains more phospholipids and lipoproteins to the filler tolerated maximum intensity elution solution can not elution, 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 requires a lot of ethanol or isopropyl alcohol, and 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 the preparation of anionic column chromatography mass production requires exchange extract hemoglobin using hemoglobin extract prepared by the method of easily 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 prepared extracting the hemoglobin solution, comprising the following steps, the steps are performed under ambient conditions of O~10 ° C:  (I) facing the packed red cells in a dialysis bag, was crushed in a hypotonic buffer to give a red blood cell homogenate;  (2) when the final saturation 0 ° C was added erythrocyte homogenate corresponding to an amount of 25% ammonium sulfate, sulfuric acid be after sufficiently dissolved ammonium centrifuged at 4000 G;  (3) step (2) when the final saturation was centrifuged and the supernatant was added 60% @ 0 ° C corresponding to an amount of ammonium sulfate until fully dissolved after centrifugation at 6000G;  (4) the step (3) the resulting protein precipitate after centrifugation was resuspended into a dialysis bag, ammonium sulfate removed by dialysis buffer;  (5) the protein solution was dialyzed buffer volume ratio of 1: 5 diluted, and then centrifuged at 10000G, particulate removal.  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 for producing a hypotonic environment dialysis manner erythrocytes broken, effectively reducing the volume of red blood cell homogenate, increasing the density of the solution, thus creating for subsequent ammonium sulfate fractionation conditions; and because of the small volume of red blood cell homogenate, 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。 Of step (2) and (3) two-step ammonium sulfate precipitation operation, prepare a solution of a different density, so that the lipoproteins and phospholipids to the hemoglobin in a different position in the centrifuge tube at the density corresponding to the centrifugal force conditions, thereby making the vast lipoproteins and heteroaryl portion of the protein fraction is 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, the hemoglobin solution after the extraction process of the present invention prepared hemoglobin extract prepared with conventional methods for anion exchange chromatography compared to the lipoprotein and lipid content of the residual pity less than 2%, by easily 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  FIG extract hemoglobin in the present invention and the conventional method of preparation for anion exchange chromatography compared, SDS-PAGE analysis result of hemoglobin in FIG control extract prepared;  FIG. in: M represents protein molecular weight standards;  C represents a hemoglobin extract prepared by a conventional method;  D represents a 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。 Drawings and embodiments of the present invention is further illustrated DETAILED DESCRIPTION  The following binding.  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  carried out at ambient temperature O~10 V sequentially the following:  (I) in the hematocrit of red blood cells an average of 60 kDa molecular weight cutoff dialysis bag was crushed to obtain erythrocyte hypotonic homogenate, dialysis the concentration of Tris base buffer is Tris-HCl buffer, after deoxygenation treatment, the buffer is 100 mM, pH value of 8. O;  (2) when the final saturation and 0 ° C of 25% corresponds to the amount of ammonium sulfate was added to 4000 G erythrocytes centrifuged homogenate, ammonium sulfate until fully dissolved; when  (3) was added to the 0 ° C in step ⑵ centrifugation supernatant obtained to a final saturation of 60% the corresponding amount of ammonium sulfate to be dissolved sufficiently centrifuged at 6000G;  (4) the step (3) the resulting precipitate was resuspended protein was transferred to an average of 60 kDa molecular weight cutoff dialysis bag to remove sulfate ammonium, dialysis buffer was deoxygenated processed Tris-HCl, NaCl buffer, Tris base buffer concentration of 55 mM, NaCl concentration of 30 mM, pH 8.5 buffer is;  (5 ) dialyzed protein solution was buffer volume ratio of 1: 5 dilution ratio and then centrifuged at 10,000 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。 Removing particulate matter, the buffer was deoxygenated treated Tris-HCl, NaCl buffer, Tris base buffer concentration of 55 mM, NaCl concentration of 30 mM, pH 8.5 buffer value.  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。  Example of the present embodiment, the hemoglobin extract was prepared using the method of the present invention, hemoglobin recovery was 87%, the residual amount of the phospholipid and lipoprotein I. 5%, by O. 45Mffl easily membrane, anion exchange column is very suitable for layers precipitation method of 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 are FIGS SDS-PAGE analysis of hemoglobin control extract prepared by the method of the present invention and the conventional method. 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, the hemoglobin extract prepared by the method of the present invention effectively reduce the protein content, and the hemoglobin content increased.