Combined reagent and method for removing RNA (Ribonucleic Acid) in plasmid purification
By using a combination reagent of CaCl2 and ammonium sulfate and specific steps, the problem of poor RNA removal effect in plasmid purification is solved, the purity and yield of the plasmid are improved, and the needs of industrial production are met.
Patent Information
- Application Number
- CN202411689936.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-01-10
AI Technical Summary
The RNA removal effect in the existing plasmid purification methods is poor, which affects the purity and yield of the plasmid. The parameter adjustment of the traditional method is complicated and it is difficult to meet the needs of industrial production.
The combined reagent of CaCl2 and ammonium sulfate is used to achieve effective removal and purification of plasmids through specific steps and concentration ratios, including resuspension, lysate, neutralization and molecular sieve chromatography steps.
The purity and yield of the plasmid are significantly improved, the cost is reduced, the needs of industrial production are met, and the effectiveness of the method is verified through the detection results.
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Figure CN119286848A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biology, and in particular to a combined reagent and method for removing RNA in plasmid purification. Background Art
[0002] Plasmid preparation is a basic technique in molecular biology experiments, mainly used to extract and purify plasmid DNA from bacterial cells. Plasmids are small, circular double-stranded DNA molecules that exist in nature and are widely found in bacteria because they can replicate themselves. They are often used as cloning vectors for the insertion and expression of foreign genes. They are used in research in many fields such as genetic engineering, gene therapy, and vaccine development. Plasmid preparation technology is also constantly innovating and improving, and it has become imperative to optimize its separation method to obtain maximum yield and purity.
[0003] Plasmid extraction methods include heat shock and alkaline lysis. Heat shock is to place bacteria in Triton-X100 and lysozyme solution, then lyse the cells in boiling water to denature the bacterial protein and chromosomal DNA, then centrifuge the denatured protein and chromosomal DNA, and then recover the plasmid DNA in the supernatant. However, the sugars in the heat shock method cannot be removed, which affects the activity of some enzymes, and the heating temperature and heat shock often cause damage to the plasmid.
[0004] The traditional alkaline lysis method for extracting plasmids is to separate covalently closed circular plasmid DNA and linear chromosomal DNA based on their topological differences. In the narrow range of pH between 12.0-12.5, the linear DNA double helix structure is unwound and denatured. Although the hydrogen bonds of the covalently closed circular plasmid DNA are broken under such conditions, the two complementary chains are entangled with each other and still tightly bound together. When the pH is restored to neutral by adding potassium acetate high salt buffer at pH 4.8, the two complementary chains of the covalently closed circular plasmid DNA remain together, so the renaturation is rapid and accurate, while the two complementary chains of the linear chromosomal DNA are completely separated from each other, and the renaturation is not so rapid and accurate. They are entangled to form a mesh structure. Through centrifugation, the chromosomal DNA is precipitated together with unstable macromolecular RNA, protein-SDS complex, etc. and removed. However, the plasmid purity and recovery rate after clarification of the liquid by this method are not high. The number and purity of the separated plasmids are greatly affected by the lysis process parameters (centrifugal force and centrifugation time). The residual RNA component in the sample is very high, and the effect of removing RNA only by molecular sieve chromatography is limited.
[0005] There is an urgent need for a method that can be used to remove RNA during plasmid purification. Summary of the invention
[0006] The object of the present invention is to provide a combined reagent and method for removing RNA in plasmid purification.
[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:
[0008] In one aspect, the present invention provides a combination reagent for removing RNA in plasmid purification, wherein the combination reagent comprises CaCl2 and ammonium sulfate.
[0009] Specifically, the concentration of CaCl2 is 5-15mM, and the concentration of ammonium sulfate is 1-5M;
[0010] Furthermore, the concentration of CaCl2 is 10mM, and the concentration of ammonium sulfate is 2M.
[0011] In another aspect, the present invention provides use of the above-mentioned combination reagent in purifying plasmids.
[0012] In another aspect, the present invention provides a method for removing RNA in plasmid purification, comprising the following steps:
[0013] (1) resuspending the bacterial strain using a resuspension solution;
[0014] (2) adding lysis solution to the resuspended bacterial solution to carry out reaction;
[0015] (3) Add neutralizing solution to adjust pH;
[0016] (4) adding CaCl2 to the neutralized solution for precipitation and centrifugation;
[0017] (5) filtering and concentrating the solution after centrifugation in step (4);
[0018] (6) Add ammonium sulfate and perform molecular sieve chromatography.
[0019] Specifically, the bacterial species described in step (1) include but are not limited to Escherichia coli DH5α.
[0020] Specifically, in step (1), the mass volume ratio of the bacterial strain to the resuspension is 1:(8-12);
[0021] Furthermore, in step (1), the mass volume ratio of the bacterial strain to the resuspension is 1:10.
[0022] Specifically, the resuspension solution includes glucose, Tris and EDTA.
[0023] Furthermore, the concentration of glucose in the resuspension is 40-60 mM; further, the concentration of glucose in the resuspension is 50 mM.
[0024] Furthermore, the Tris in the resuspension is a combination of Tris with different pH values; and further, the pH values of the Tris are 6.8 and 11, respectively.
[0025] According to some embodiments of the present invention, the Tris (pH 6.8): Tris (pH 11) = 4:1.
[0026] Specifically, the concentration of Tris is 20-30 mM; further, the concentration of Tris is 25 mM.
[0027] Specifically, the concentration of EDTA is 5-15 mM; further, the concentration of EDTA is 10 mM.
[0028] Specifically, the pH of the resuspension is 8.0.
[0029] According to some embodiments of the present invention, the resuspension solution comprises 50 mM glucose, 20 mM Tris-HCl, 5 mM Tris and 10 mM EDTA.
[0030] Specifically, step (1) further includes a stirring step after adding the resuspension liquid.
[0031] Furthermore, the stirring speed is 500 rpm and the stirring time is 40 min.
[0032] Specifically, the volume ratio of the lysate added in step (2) to the resuspension solution is 1:(1-3);
[0033] Furthermore, the volume ratio of the lysate added in step (2) to the resuspension solution is 1:1.
[0034] Specifically, the reaction time in step (2) is 5-10 min; further, the reaction time in step (2) is 6 min.
[0035] Specifically, the lysis solution in step (2) includes NaOH and SDS.
[0036] Furthermore, the lysate includes 0.1-0.5M NaOH and 1-5% SDS;
[0037] Furthermore, the lysate includes 0.2 M NaOH and 1% SDS.
[0038] Specifically, in step (3), the volume ratio of the neutralization solution to the resuspension solution is 1:(1-3);
[0039] Furthermore, in step (3), the volume ratio of the neutralization solution to the resuspension solution is 1:1.
[0040] Specifically, the neutralizing solution in step (3) includes potassium acetate and acetic acid.
[0041] Further, the neutralization solution includes 1-5M potassium acetate and 1-5M acetic acid;
[0042] Furthermore, the neutralization solution includes 3M potassium acetate and 2M acetic acid.
[0043] Specifically, the concentration of CaCl2 in step (4) is 5-15 mM; further, the concentration of CaCl2 in step (4) is 10 mM.
[0044] Specifically, the centrifugal speed in step (4) is 3000-5000 rpm, and the centrifugal time is 20-50 min;
[0045] Furthermore, in step (4), the centrifugal speed is 4000 rpm and the centrifugal time is 40 min.
[0046] Specifically, in step (5), a 0.45 / 0.22 um capsule filter is used for filtration, and the filtration pressure is controlled to be less than 0.5 bar.
[0047] Specifically, in step (5), a Cobite TFF system was used for concentration operation, during which the TMP was always controlled at 3psi-3.5psi and the flow rate was 212ml / min.
[0048] Specifically, the concentration of ammonium sulfate in step (6) is 1-5M; further, the concentration of ammonium sulfate is 2M.
[0049] Specifically, the specific operation of molecular sieve chromatography in step (6) is: use Buffer I to balance the chromatography column 150 ml, flow rate 20 ml / min, flow path Forword, loading speed: 10 ml / min, and use Buffer I for post-balancing after loading.
[0050] Specifically, the Buffer I described in step (6) includes ammonium sulfate, EDTA and Tris.
[0051] Furthermore, the Buffer I described in step (6) includes 1-3 M ammonium sulfate, 8-12 M EDTA and 80-120 mM Tris.
[0052] Furthermore, the Buffer I described in step (6) includes 2.1 M ammonium sulfate, 10 M EDTA and 100 mM Tris.
[0053] Furthermore, the Tris in the Buffer I is a combination of Tris with different pH values; and further, the pH values of the Tris are 6.8 and 11, respectively.
[0054] According to some embodiments of the present invention, the Tris (pH 6.8): Tris (pH 11) = 6:4.
[0055] The beneficial effects of the present invention are:
[0056] The present invention provides a combined reagent and method for removing RNA in plasmid purification. The optimized purification method can effectively reduce costs, improve plasmid purity and yield, and can achieve purification of plasmids of different volumes, which can meet the needs of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Figure 1 The results are shown in Example 1.
[0058] Figure 2 This is the test result in Comparative Example 1.
[0059] Figure 3 This is a graph showing the total amount of plasmid and yield in Comparative Example 3.
[0060] Figure 4 It is the chromatogram of Comparative Example 3. DETAILED DESCRIPTION
[0061] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.
[0062] Example 1
[0063] 1 Experimental methods
[0064] 1.1 Solution formulation
[0065] 1.1.1 Resuspension
[0066] Table 1
[0067]
[0068]
[0069] 1.1.2 Lysis buffer
[0070] Table 2
[0071]
[0072] 1.1.3 Neutralizing liquid
[0073] Table 3
[0074]
[0075] 1.1.4 Buffer I
[0076] Table 4
[0077]
[0078] Resuspend the strain (DH5α): Use the resuspending solution to resuspend 66 g of the strain, add 660 ml of the resuspending solution at a mass volume ratio of 1:10, put the stirring bar into the bottle, place it on the magnetic stirrer, set the stirring speed to 500 rpm, and stir for 40 minutes, until there are no particles visible to the naked eye.
[0079] Lysis: Add the same volume (660 ml) of lysis buffer (containing 10 mM CaCl2) to the resuspended bacterial solution and react for 6 minutes. Mix gently to prevent genomic DNA breakage.
[0080] Neutralization: The pH of the solution was lowered by adding 660 ml of neutralizing solution (containing 10 mM CaCl2).
[0081] Salt precipitation of RNA: Add CaCl2 (10 mM) solution to the neutralized solution obtained by centrifugation for precipitation, and then centrifuge at a speed of 4000 rpm for 40 min.
[0082] Ultrafiltration concentration and liquid exchange: After centrifugation, use a 0.45 / 0.22um capsule filter for filtration, control the filtration pressure to less than 0.5bar, and use the Cobite TFF system for concentration. During the operation, always control the TMP: 3psi (<3.5psi), and the flow rate is 212ml / min.
[0083] Molecular sieve chromatography: Use BufferⅠ to balance the chromatography column 150ml, flow rate 20ml / min, flow path Forword, loading speed: 10ml / min. After loading, use BufferⅠ to balance and flush out the sample peak.
[0084] Experimental results:
[0085] 1. CaCl2 and ammonium sulfate group
[0086] The CaCl2 concentration was 10mM. 66g of wet bacteria were dissolved in 1L of resuspension solution and then divided into two groups, respectively recorded as Group A and Group B. The lysis and neutralization processes of Group A and Group B were consistent. During the salt precipitation of RNA, 10mM CaCl2 was added to Group A, but not to Group B. Before molecular sieve chromatography, 2M ammonium sulfate was added to the concentrate of Group A for chromatography, but no ammonium sulfate was added to Group B. The final data were detected using a Thermo Fisher microplate reader as follows:
[0087] Table 5
[0088]
[0089] The results are as follows Figure 1 Peak 123 is group A, 1 is sample peak, 2 is impurity peak, 3 is CIP peak, peak 456 is group B, 4 is sample peak, 5 is impurity peak, 6 is CIP peak. According to the spectrum, the RNA peak of the group (group A) with the addition of CaCl2 and ammonium sulfate is greatly reduced, and the total amount of plasmid is higher than that of the control group. Therefore, this method can indeed stably and reliably remove a large amount of RNA and ensure a certain plasmid yield.
[0090] Comparative Example 1
[0091] 1.1 NaHCO3 group: After neutralization, the cells were divided into three groups. During the RNA precipitation step, 0.1 M NaHCO3 was added to one group, 1 M NaHCO3 was added to the other group, and the last group was not added as the control group (NX240516, 6FF-240517).
[0092] 1.1.1 Ultrafiltration concentration and liquid replacement (concentration and liquid replacement are the same as in Example 1)
[0093] Table 6
[0094]
[0095] 1.2 The molecular sieve chromatography step is the same as in Example 1.
[0096] Table 7
[0097]
[0098]
[0099] According to the above data, Figure 2 ,from Figure 2 It can be seen that the addition of NaHCO3 did not increase the plasmid yield, and the total amount of plasmid was much lower than that of the group without NaHCO3.
[0100] Comparative Example 2 CaCl2 Group
[0101] The only difference from Example 1 is that Comparative Example 2 does not include the molecular sieve chromatography step, and the rest is the same.
[0102] Ultrafiltration concentration and liquid replacement:
[0103] Table 8
[0104]
[0105] According to the detection, the loss of DNA in the CaCL2 group was too much.
[0106] Comparative Example 3 Ammonium sulfate group
[0107] The only difference compared with Example 1 is that the step of adding CaCl2 (10 mM) solution for precipitation treatment is not included, and the rest are the same.
[0108] 1Ultrafiltration concentration and liquid replacement
[0109] Table 9
[0110]
[0111] 2 Molecular sieve chromatography
[0112] Table 10
[0113]
[0114] According to the above data, Figure 3 As can be seen from the figure, the plasmid yield was slightly improved by adding ammonium sulfate. The chromatogram is shown in Figure 4 As shown, from Figure 4 It can be seen that the impurity peak (4) is significantly reduced in the molecular sieve chromatography after the addition of ammonium sulfate.
[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A combination reagent for removing RNA in plasmid purification, characterized in that: The combined reagent includes CaCl2 and ammonium sulfate.
2. The combined reagent according to claim 1, characterized in that: The concentration of CaCl2 is 5-15mM, and the concentration of ammonium sulfate is 1-5M.
3. Use of the combined reagent according to any one of claims 1 to 2 in purifying plasmids.
4. A method for removing RNA in plasmid purification, characterized in that: The following steps are involved: (1) resuspending the bacterial strain using a resuspension solution; (2) adding lysis solution to the resuspended bacterial solution to carry out reaction; (3) Add neutralizing solution to adjust pH; (4) adding CaCl2 to the neutralized solution for precipitation and centrifugation; (5) filtering and concentrating the solution after centrifugation in step (4); (6) Add ammonium sulfate and perform molecular sieve chromatography.
5. The method according to claim 4, characterized in that In step (1), the mass volume ratio of the bacterial strain to the resuspension is 1:(8-12); the resuspension comprises glucose, Tris and EDTA.
6. The method according to claim 4, characterized in that The volume ratio of the lysis solution added in step (2) to the resuspension solution is 1:(1-3); the lysis solution in step (2) includes NaOH and SDS.
7. The method according to claim 4, characterized in that The volume ratio of the neutralization solution to the resuspension solution in step (3) is 1:(1-3); the neutralization solution in step (3) includes potassium acetate and acetic acid.
8. The method according to claim 4, characterized in that The concentration of CaCl2 in step (4) is 5-15 mM; the concentration of ammonium sulfate in step (6) is 1-5 M.
9. The method according to claim 4, characterized in that The concentration of CaCl2 in step (4) is 10 mM; the concentration of ammonium sulfate in step (6) is 2 M.
10. The method according to claim 4, characterized in that The molecular sieve chromatography in step (6) includes using a Buffer I equilibrium chromatography column, wherein the Buffer I includes ammonium sulfate, EDTA and Tris.