CN115074355A

Movatterモバイル変換

Info

Publication number: CN115074355A
Application number: CN202210807826.3A
Authority: CN
Inventors: 郝好超
Original assignee: Wuxi ATU Co Ltd
Current assignee: Wuxi ATU Co Ltd
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2022-09-20

Abstract

Translated fromChinese

本发明涉及质粒碱裂解技术领域，特别是涉及一种提高质粒碱裂解效率的方法，包括：将菌体和第一裂解液混合重悬处理形成菌体重悬液，所述菌体重悬液的菌体浓度为40～80g/kg；将菌体重悬液与第二裂解液按照体积比为1：(0.9～1.1)混合，碱裂解处理至少6min，即得菌体碱裂解液；将第二裂解液与第三裂解液按照体积比为1：(1～2)混合，中和处理，即可。本发明可有效提高大规模质粒裂解过程中操作的便易性，提高碱裂解效率，且质粒质量不受影响。

The invention relates to the technical field of plasmid alkaline lysis, in particular to a method for improving the efficiency of plasmid alkaline lysis, comprising: mixing and resuspension of bacterial cells and a first lysing solution to form a bacterial re-suspension; The cell concentration is 40-80 g/kg; the bacterial suspension and the second lysing solution are mixed according to the volume ratio of 1: (0.9-1.1), and the alkaline lysing treatment is performed for at least 6 minutes to obtain the bacterial cell alkaline lysing solution; the second lysing solution is The solution and the third lysing solution are mixed according to the volume ratio of 1: (1-2), and then neutralized. The invention can effectively improve the ease of operation in the large-scale plasmid splitting process, improve the alkali splitting efficiency, and the plasmid quality is not affected.

Description

Translated fromChinese

一种提高质粒碱裂解效率的方法A kind of method for improving plasmid alkaline cleavage efficiency

技术领域technical field

本发明涉及质粒碱裂解技术领域，特别是涉及一种提高质粒碱裂解效率的方法。The invention relates to the technical field of plasmid alkaline lysis, in particular to a method for improving the efficiency of plasmid alkaline lysis.

背景技术Background technique

在质粒生产过程中，碱裂解是为了裂解大肠杆菌细胞，释放质粒，从而为下游纯化提供内含质粒的细胞裂解液。碱裂解的效率对整个工艺中质粒的回收率有很大的影响，其中菌浓、裂解时间是关键调控点。During plasmid production, alkaline lysis is used to lyse E. coli cells, releasing the plasmid, thereby providing a plasmid-containing cell lysate for downstream purification. The efficiency of alkaline lysis has a great influence on the recovery rate of plasmids in the whole process, among which bacterial concentration and lysis time are the key control points.

行业内传统碱裂解过程，裂解液的具体成分如表1所示：In the traditional alkaline cracking process in the industry, the specific components of the cracking solution are shown in Table 1:

溶液名称Solution name溶液成份Solution composition裂解液1Lysate 150mM Tris,20mM glucose,10mM EDTA,pH 8.050mM Tris, 20mM glucose, 10mM EDTA, pH 8.0裂解液2Lysate 20.2M NaOH,1％SDS0.2M NaOH, 1% SDS裂解液3Lysate 33M KAc,2M HAc3M KAc, 2M HAc

表1Table 1

基于表格1所示的裂解液，按照传统碱裂解菌浓90g/kg，且裂解液1：裂解液2：裂解液3＝1：2：1.5(体积比)进行处理。虽然上述处理工艺可以小规模操作且简单便宜，但是放大规模后，可操作性差，可控性差；其中，裂解液2的处理时间为5min会导致操作时间少，工艺可控性差，风险高。传统碱裂解效率一般维持在30％-50％，即理论上菌体内含100mg质粒，碱裂解后只能释放30-50mg质粒，存在碱裂解效率低的问题。Based on the lysing solution shown in Table 1, the traditional alkaline lysing bacteria concentration was 90 g/kg, and the lysing solution 1: the lysing solution 2: the lysing solution 3=1:2:1.5 (volume ratio) for treatment. Although the above-mentioned treatment process can be operated on a small scale and is simple and cheap, after scale-up, the operability and controllability are poor; among them, the treatment time oflysate 2 is 5min, which will lead to less operation time, poor process controllability and high risk. The traditional alkaline lysis efficiency is generally maintained at 30%-50%, that is, theoretically the bacteria contain 100 mg of plasmid, and only 30-50 mg of plasmid can be released after alkaline lysis, which has the problem of low alkaline lysis efficiency.

发明内容SUMMARY OF THE INVENTION

鉴于以上所述现有技术的缺点，本发明的目的在于提供一种提高质粒碱裂解效率的方法，用于解决现有技术中质粒碱裂解效率低的问题。本发明可有效提高大规模质粒裂解过程中操作的便易性，提高碱裂解效率，且质粒质量不受影响。In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for improving the efficiency of alkaline lysis of plasmids, so as to solve the problem of low efficiency of alkaline lysis of plasmids in the prior art. The invention can effectively improve the ease of operation in the large-scale plasmid splitting process, improve the alkali splitting efficiency, and the plasmid quality is not affected.

为实现上述目的及其他相关目的，In order to achieve the above and other related purposes,

本发明的第一方面，提供一种提高质粒碱裂解效率的方法，包括如下步骤：A first aspect of the present invention provides a method for improving the efficiency of plasmid alkali lysis, comprising the steps of:

步骤一、将菌体和第一裂解液混合，重悬处理形成菌体重悬液，所述菌体重悬液的菌体浓度为40～80g/kg；Step 1, mixing the thalline and the first lysate, and resuspending to form a thalline suspension, the thalline concentration of the said thalline suspension is 40～80g/kg;

步骤二、将菌体重悬液与第二裂解液按照体积比为1：(0.9～1.1)混合，碱裂解处理至少6min，即得菌体碱裂解液；Step 2: Mix the bacterial resuspended liquid with the second lysate according to a volume ratio of 1: (0.9 to 1.1), and perform an alkaline lysis treatment for at least 6 minutes to obtain a bacterial cell alkaline lysate;

步骤三、将第二裂解液与第三裂解液按照体积比为1：(1～2)混合，中和处理，即可。Step 3: Mix the second lysate and the third lysate according to a volume ratio of 1:(1-2), and then neutralize them.

于本发明的一实施例中，所述步骤一中菌体的质量与第一裂解液的体积比值为1：(9～24)。In an embodiment of the present invention, the ratio of the mass of the bacterial cells to the volume of the first lysate in the first step is 1:(9-24).

于本发明的一实施例中，所述步骤一中菌体重悬液的菌体浓度为40～60g/kg。In an embodiment of the present invention, the bacterial cell concentration of the bacterial resuspended liquid in thestep 1 is 40-60 g/kg.

于本发明的一实施例中，所述步骤一中重悬处理的搅拌速度为300～600rpm，重悬时间为30～60min。In an embodiment of the present invention, the stirring speed of the resuspension treatment in the first step is 300-600 rpm, and the resuspension time is 30-60 min.

于本发明的一实施例中，所述步骤二中菌体重悬液与第二裂解液的体积比为1：(0.98～1.02)。In an embodiment of the present invention, the volume ratio of the bacterial suspension to the second lysate in the second step is 1:(0.98-1.02).

于本发明的一实施例中，所述步骤二中碱裂解处理的搅拌速度为30～100rpm，碱裂解时间为6～20min。In an embodiment of the present invention, the stirring speed of the alkali cracking treatment in the second step is 30-100 rpm, and the alkali cracking time is 6-20 min.

于本发明的一实施例中，所述步骤三中第二裂解液与第三裂解液按照体积比为1：(1.4～1.6)。In an embodiment of the present invention, the volume ratio of the second lysing solution to the third lysing solution in the third step is 1:(1.4-1.6).

于本发明的一实施例中，所述中和处理的搅拌速度为30～100rpm，中和时间为8～12min。In an embodiment of the present invention, the stirring speed of the neutralization treatment is 30-100 rpm, and the neutralization time is 8-12 min.

于本发明的一实施例中，所述第一裂解液包括如下组分：80～120mM的氨基丁三醇，40～60mM的葡萄糖，8～12mM的乙二胺四乙酸；In an embodiment of the present invention, the first lysing solution includes the following components: 80-120 mM tromethamine, 40-60 mM glucose, and 8-12 mM EDTA;

所述第二裂解液包括如下组分：0.25～0.35M的氢氧化钠，1.8～2.2％的SDS；The second lysis solution includes the following components: 0.25-0.35M sodium hydroxide, 1.8-2.2% SDS;

所述第三裂解液包括如下组分：2.2～2.8M的醋酸钾，2.2～2.8M的醋酸。The third lysis solution includes the following components: 2.2-2.8M potassium acetate, 2.2-2.8M acetic acid.

于本发明的一实施例中，所述第一裂解液包括如下组分：95～105mM的氨基丁三醇，45～55mM的葡萄糖，9～11mM的乙二胺四乙酸；In an embodiment of the present invention, the first lysis solution includes the following components: 95-105 mM tromethamine, 45-55 mM glucose, and 9-11 mM EDTA;

所述第二裂解液包括如下组分：0.29～0.31M的氢氧化钠，1.95～2.05％的SDS；The second lysis solution includes the following components: 0.29-0.31M sodium hydroxide, 1.95-2.05% SDS;

所述第三裂解液包括如下组分：2.45～2.55M的醋酸钾，2.45～2.55M的醋酸。The third lysis solution includes the following components: 2.45-2.55M potassium acetate, 2.45-2.55M acetic acid.

如上所述，本发明的一种提高质粒碱裂解效率的方法，具有以下有益效果：本发明可有效提高大规模质粒裂解过程中操作的便易性，提高碱裂解效率，有效提高碱裂解效率至60％～90％，质粒质量不受影响，便于工艺放大性、自动化性、可控性等。As mentioned above, a method for improving the efficiency of plasmid alkali lysis of the present invention has the following beneficial effects: the present invention can effectively improve the ease of operation in the large-scale plasmid lysis process, improve the efficiency of alkali lysis, and effectively improve the efficiency of alkali lysis to 60% to 90%, the quality of the plasmid is not affected, and it is convenient for process amplification, automation and controllability.

附图说明Description of drawings

图1为本发明实施例1～6的裂解效率曲线图。FIG. 1 is a graph showing the cracking efficiency of Examples 1 to 6 of the present invention.

图2为本发明实施例7～10的裂解效率曲线图。Fig. 2 is a graph showing the cracking efficiency of Examples 7-10 of the present invention.

图3为本发明实施例11～18的裂解效率曲线图。3 is a graph showing the cracking efficiency of Examples 11 to 18 of the present invention.

具体实施方式Detailed ways

以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。The embodiments of the present invention are described below by specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

实施例1Example 1

一种提高质粒碱裂解效率的方法，包括如下步骤：A method for improving the efficiency of plasmid alkali lysis, comprising the steps:

步骤一、用天平称取目标菌体，按照菌体量(g)：第一裂解液(mL)＝1：9～24的质量体积比混合后形成菌体重悬液(菌体浓度为40g/kg)，再进行菌体重悬，搅拌转速400rpm，重悬40min，至无肉眼可见明显颗粒即可；Step 1. Weigh the target thalline with a balance, and form a thalline suspension after mixing according to the mass of the thalline (g): the first lysate (mL) = 1:9 to 24 (the thalline concentration is 40 g/ kg), resuspend the bacteria again, stirring at 400rpm, and resuspend for 40min, until no obvious particles are visible to the naked eye;

步骤二、菌体重悬液：第二裂解液的体积比为1：1，分别通过蠕动泵中的双管道，等流速的混合在一起形成菌体碱裂解液，进行碱裂解，控制泵速，保证裂解时间维持在12min，混合过程搅拌转速50rpm；Step 2. Bacterial re-suspension: the volume ratio of the second lysate is 1:1, respectively, through the double pipelines in the peristaltic pump, and the equal flow rates are mixed together to form the bacterium alkaline lysate, and the alkaline lysate is performed to control the pump speed. Ensure that the cracking time is maintained at 12min, and the stirring speed of the mixing process is 50rpm;

步骤三、第二裂解液：第三裂解液的体积比为1：1.5，使用蠕动泵的单管道加入第三裂解液进行中和，控制泵速，保证中和时间维持在10min，中和过程搅拌转速50rpm；Step 3. The second lysate: the volume ratio of the third lysate is 1:1.5, use the single pipe of the peristaltic pump to add the third lysate for neutralization, control the pump speed, and ensure that the neutralization time is maintained at 10min. Stirring speed 50rpm;

步骤四、碱裂解结束后，进行澄清过滤等。Step 4. After the alkali cracking is completed, clarification and filtration are carried out.

实施例2～6Examples 2 to 6

实施例2～6与实施例1的区别在于菌体浓度不同，实施例1～6的菌体浓度如表格2所示：The difference between Examples 2 to 6 and Example 1 is that the bacterial concentration is different. The bacterial concentration of Examples 1 to 6 is shown in Table 2:

实施例Example菌体浓度/(g/kg)Bacterial concentration/(g/kg)裂解效率/％Lysis efficiency/%114040909022505086863360608585447070707055808063636690903535

表2Table 2

表格2中的裂解效率曲线图如图1所示。从图1和表格2中可以看出，同一条件下，40-90g/kg菌浓范围内，碱裂解菌浓越高，裂解效率越低。碱裂解菌浓设置在40-80g/kg时，裂解效率高于60％；且菌体浓度优选为40-60g/kg。The cleavage efficiency graph in Table 2 is shown in Figure 1 . As can be seen from Figure 1 and Table 2, under the same conditions, within the bacterial concentration range of 40-90 g/kg, the higher the concentration of alkaline lysing bacteria, the lower the lysis efficiency. When the concentration of alkaline lysing bacteria is set at 40-80 g/kg, the lysis efficiency is higher than 60%; and the concentration of bacteria is preferably 40-60 g/kg.

实施例7Example 7

步骤一、用天平称取目标菌体，按照菌体量(g)：第一裂解液(mL)＝1：9～24的质量体积比混合后形成菌体重悬液(菌体浓度为70g/kg)，再进行菌体重悬，搅拌转速400rpm，重悬40min，至无肉眼可见明显颗粒即可；Step 1. Weigh the target thalline with a balance, and form a thalli suspension after mixing according to the thalline amount (g): the first lysate (mL) = 1:9 to 24 mass-volume ratio (the thalline concentration is 70 g/ kg), resuspend the bacteria again, stirring at 400rpm, and resuspend for 40min, until no obvious particles are visible to the naked eye;

步骤三、菌体碱裂解液：第三裂解液的体积比为1：1，使用蠕动泵的单管道加入第三裂解液进行中和，控制泵速，保证中和时间维持在10min，中和过程搅拌转速50rpm；Step 3. Bacterial alkaline lysate: the volume ratio of the third lysate is 1:1. Use the single pipe of the peristaltic pump to add the third lysate for neutralization, control the pump speed, and ensure that the neutralization time is maintained at 10min. Process stirring speed 50rpm;

实施例8～10Examples 8 to 10

实施例8～10与实施例7的区别在于体积比不同，实施例7～10的体积比如表格3所示：The difference between Examples 8 to 10 and Example 7 is that the volume ratio is different. The volume ratios of Examples 7 to 10 are shown in Table 3:

表3table 3

表格3中的裂解效率曲线图如图2所示。从图2和表格3中可以看出，同一条件下，碱裂解缓冲液体积比例(1：1：1～2)相比传统碱裂解缓冲液体积比例(1：2：1.5)裂解效率比较高。The cleavage efficiency graph in Table 3 is shown in FIG. 2 . As can be seen from Figure 2 and Table 3, under the same conditions, the volume ratio of alkaline lysis buffer (1:1:1-2) is more efficient than the traditional volume ratio of alkaline lysis buffer (1:2:1.5) .

实施例11Example 11

步骤二、菌体重悬液：第二裂解液的体积比为1：1，分别通过蠕动泵中的双管道，等流速的混合在一起形成菌体碱裂解液，进行碱裂解，控制泵速，保证裂解时间维持在6min，混合过程搅拌转速50rpm；Step 2. Bacterial re-suspension: the volume ratio of the second lysate is 1:1, respectively, through the double pipelines in the peristaltic pump, and the equal flow rates are mixed together to form the bacterium alkaline lysate, and the alkaline lysate is performed to control the pump speed. Ensure that the cracking time is maintained at 6min, and the stirring speed of the mixing process is 50rpm;

步骤三、菌体碱裂解液：第三裂解液的体积比为1：1.5，使用蠕动泵的单管道加入第三裂解液进行中和，控制泵速，保证中和时间维持在10min，中和过程搅拌转速50rpm；Step 3. Bacterial alkaline lysate: the volume ratio of the third lysate is 1:1.5. Use the single channel of the peristaltic pump to add the third lysate for neutralization, control the pump speed, and ensure that the neutralization time is maintained at 10min. Process stirring speed 50rpm;

实施例12～18Examples 12 to 18

实施例12～18与实施例11的区别在于碱裂解时间不同，实施例11～18的碱裂解时间如表格4所示：The difference between Examples 12 to 18 and Example 11 is that the alkali cracking time is different. The alkali cracking time of Examples 11 to 18 is shown in Table 4:

表4Table 4

表格4中的裂解效率曲线图如图3所示。从图3和表格4中可以看出，同一条件下，随着裂解时间的增加(20min以内)，裂解效率呈现增长状。The cleavage efficiency graph in Table 4 is shown in FIG. 3 . As can be seen from Figure 3 and Table 4, under the same conditions, with the increase of the cracking time (within 20min), the cracking efficiency showed an increasing shape.

上述实施例1～18的裂解液的组分如表格5所示：The components of the lysates of the above Examples 1 to 18 are shown in Table 5:

表5table 5

综上所述，本发明可有效提高大规模质粒裂解过程中操作的便易性，提高碱裂解效率，进而提高质粒产量。所以，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。To sum up, the present invention can effectively improve the ease of operation in the large-scale plasmid lysis process, improve the efficiency of alkaline lysis, and thereby improve the yield of plasmids. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims

Translated fromChinese

1.一种提高质粒碱裂解效率的方法，其特征在于，包括如下步骤：1. a method for improving plasmid alkali cleavage efficiency, is characterized in that, comprises the steps:

2.根据权利要求1所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤一中菌体的质量与第一裂解液的体积比值为1：(9～24)。2 . The method for improving the efficiency of alkaline lysis of plasmids according to claim 1 , wherein in the step 1, the mass ratio of the bacterial cells to the volume of the first lysate is 1: (9-24). 3 .

3.根据权利要求2所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤一中菌体重悬液的菌体浓度为40～60g/kg。3 . The method for improving the efficiency of alkaline lysis of plasmids according to claim 2 , wherein in the step 1, the bacterial cell concentration of the bacterial resuspended liquid is 40-60 g/kg. 4 .

4.根据权利要求1～3任一项所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤一中重悬处理的搅拌速度为300～600rpm，重悬时间为30～60min。4. a kind of method that improves the efficiency of plasmid alkali lysis according to any one of claim 1～3, it is characterized in that: in described step 1, the stirring speed of resuspension treatment is 300～600rpm, and the resuspension time is 30～600rpm. 60min.

5.根据权利要求1所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤二中菌体重悬液与第二裂解液的体积比为1：(0.98～1.02)。5 . The method for improving the efficiency of plasmid alkaline lysis according to claim 1 , wherein the volume ratio of the bacterial suspension to the second lysis solution in the second step is 1:(0.98～1.02). 6 .

6.根据权利要求1或5所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤二中碱裂解处理的搅拌速度为30～100rpm，碱裂解时间为6～20min。6. a kind of method that improves plasmid alkali cracking efficiency according to claim 1 or 5, is characterized in that: in described step 2, the stirring speed of alkali cracking treatment is 30～100rpm, and alkali cracking time is 6～20min.

7.根据权利要求1所述的一种提高质粒碱裂解效率的方法，其特征在于：所述步骤三中第二裂解液与第三裂解液按照体积比为1：(1.4～1.6)。7 . The method for improving the efficiency of plasmid alkaline lysis according to claim 1 , wherein the second lysate and the third lysate are in a volume ratio of 1:(1.4～1.6) in the step 3. 8 .

8.根据权利要求1或7所述的一种提高质粒碱裂解效率的方法，其特征在于：所述中和处理的搅拌速度为30～100rpm，中和时间为8～12min。8. The method for improving the efficiency of plasmid alkaline lysis according to claim 1 or 7, wherein the stirring speed of the neutralization treatment is 30-100 rpm, and the neutralization time is 8-12 min.

9.根据权利要求1所述的一种提高质粒碱裂解效率的方法，其特征在于：所述第一裂解液包括如下组分：80～120mM的氨基丁三醇，40～60mM的葡萄糖，8～12mM的乙二胺四乙酸；9. The method for improving the efficiency of plasmid alkaline lysis according to claim 1, wherein the first lysis solution comprises the following components: 80-120 mM tromethamine, 40-60 mM glucose, 8 ~12 mM EDTA;

10.根据权利要求1或9所述的一种提高质粒碱裂解效率的方法，其特征在于：所述第一裂解液包括如下组分：95～105mM的氨基丁三醇，45～55mM的葡萄糖，9～11mM的乙二胺四乙酸；10. The method for improving the efficiency of plasmid alkaline lysis according to claim 1 or 9, wherein the first lysis solution comprises the following components: 95-105mM tromethamine, 45-55mM glucose , 9～11mM EDTA;