Virus-like vector lysis buffer and virus-like vector lysis method thereof

Abstract

The invention provides a virus-like vector lysis solution and a virus-like vector lysis method. The virus-like vector lysis solution comprises Tris, EDTA (Ethylene Diamine Tetraacetic Acid), NaCl, TritonX-100, sodium deoxycholate, SDS (Sodium Dodecyl Sulfate), Tween 20 and a protease inhibitor mixture. Cas9 protein is wrapped in the viroid vector; the acquisition method of the viroid vector comprises the step of transfecting plasmids containing a structural gene sequence for expressing the viroid vector into virus producing cells to obtain the viroid vector. The cracking method of the viroid vector comprises the following steps: S1, using: carrying out cracking treatment on a viroid vector solution by using the cracking solution; and S2, performing Cas9 protein content detection on substances released after the carrier is cracked. The invention establishes a formula of the special lysate for VLP and a corresponding cracking method, and ensures high cracking efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a virus-like vector lysis solution and a virus-like vector lysis method thereof. Background Art

[0002] The present invention relates to a method for lysing VLPs. The Chinese patent with publication number CN110684804A discloses a novel virus-like vector for delivering exogenous RNP and a preparation method thereof, wherein the envelope of the vector is an envelope containing vesicular stomatitis virus envelope glycoprotein (VSV-G), which encapsulates Sp Cas9 protein (Cas9 protein) and the sequence gRNA sequence targeting the target site can perform site-specific cutting on the target site. A method suitable for detecting the content of Cas9 protein in intermediates in each production link of virus-like vectors is needed to evaluate the efficiency of vector packaging and ensure the effectiveness of the product throughout the process.

[0003] However, there is no kit on the market that can be used directly for this test, nor is there any research on viral vector lysis. Conventional commercial lysis buffers cannot fully lyse the VLP shell structure (e.g. Figure 6 ), so that Cas9 in the vector is released, and there is a long lysis time and the lysis conditions are not conducive to the operation. For example, the conventional method for lysing cells or virus products is mainly to use a self-prepared 1 ~ 2% concentration TritonX-100 solution for a long lysis operation (depending on the characteristics of the project, the lysis time may be more than 1 hour), or to use a commercial RIPA lysis solution, and then add protease inhibitors to perform low-temperature lysis operations. Therefore, it is necessary to develop a special lysis solution suitable for virus-like vector products, as well as corresponding lysis conditions.

[0004] SDS (sodium dodecyl sulfate) is an anionic detergent, commonly used in routine operations that require denatured proteins, such as SDS-PAGE electrophoresis, bacteria, and cell lysis. As a denaturant and solubilizing agent for proteins, it can destroy intra- and inter-molecular hydrogen bonds, cause molecular unfolding, and destroy the secondary and tertiary structures of proteins. Triton X-100 is a non-ionic surfactant and emulsifier, commonly used to dissolve proteins or for membrane recovery under mild non-denaturing conditions. Triton X-100 has a non-polar hydrophilic head, which has weak interference with protein-protein interactions and is more beneficial for separating functional protein complexes. Sodium deoxycholate is an anionic surfactant that can effectively lyse cells and viruses and destroy protein interactions. It is often used to prepare cell lysates and adipose tissue lysates, and is also used for the extraction of membrane proteins, nucleoproteins, and lipoproteins. Tween 20 is a non-ionic surfactant, usually used to block non-specific protein interactions in solid-phase immunocytochemistry reactions such as immunoblotting.

[0005] The present invention is mainly based on the formula of RIPA lysis solution, optimizes or adds the concentration of the above-mentioned components with lysis effect, and develops a VLP special lysis solution formula and lysis method. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a virus-like vector lysis solution and a virus-like vector lysis method and a virus-like vector lysis method.

[0007] The present invention is achieved through the following technical solutions: <First aspect> The present invention provides a virus-like vector lysate, which is prepared from raw materials containing the following components and amounts: Tris 50-60 mM, EDTA 10-20 mM, NaCl150-200mM, TritonX-1001-3vol% Sodium deoxycholate 0.5-1 wt%, SDS0.5- 0.1 vol%, Tween 201-2 vol% Protease inhibitor cocktail 1-2 vol%.

[0008] Preferably, the virus-like vector lysate is prepared from raw materials containing the following components and amounts: Tris 50 mM, EDTA 20 mM, NaCl 150 mM, TritonX-1002 vol%, Sodium deoxycholate 1 wt%, SDS0.1 vol%, Tween 202 vol%, Protease inhibitor cocktail 1 vol%.

[0009] The protease inhibitor cocktail contains 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64 and 1 mM Leupeptin, and the solvent is high-purity DMSO.

[0010] The Cas9 protein is encapsulated in the virus-like vector.

[0011] <Second Aspect> The present invention provides the use of the above lysate in detecting virus-like vectors.

[0012] The Cas9 protein is encapsulated in the virus-like vector; and the detection includes detecting the content of the Cas9 protein in the virus-like vector.

[0013] <Third Aspect> The present invention provides a method for cleaving a virus-like vector, wherein the virus-like vector contains a Cas9 protein, and the cleavage method comprises the following steps: S1, using the lysis solution as described above to lyse the virus-like vector solution; S2. Detect the Cas9 protein content in the substances released after vector lysis.

[0014] In step S1, the method for obtaining the virus-like vector liquid includes: transfecting a plasmid containing a gene sequence expressing a virus-like vector structure into a virus production cell to obtain the liquid.

[0015] As one embodiment of the present invention, the plasmid containing the gene sequence of the viral-like vector structure includes pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 4.

[0016] The molar ratio of pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 4 is 1:1:1:1:2:2.

[0017] As another embodiment of the present invention, the plasmid containing the gene sequence of the viral-like vector structure includes pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 5.

[0018] The molar ratio of pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 5 in the plasmid is 1:1:1:1:2:2.

[0019] The virus-like vector liquid includes a crude virus-like vector liquid, or a concentrated virus-like vector liquid obtained by centrifugal filtration, concentration and liquid replacement, and chromatography purification of the crude virus-like vector liquid.

[0020] The method of changing the liquid includes but is not limited to: using ultrafiltration tubes, ultrafiltration membrane packages or hollow fiber columns for ultrafiltration concentration and liquid change.

[0021] The chromatography purification method includes but is not limited to: purification by anion chromatography, cation chromatography, affinity chromatography, molecular sieve chromatography or composite chromatography.

[0022] The p24 concentration of the crude venom of the virus-like vector is 200-400 ng / mL.

[0023] The p24 concentration of the virus-like vector concentrate is 1000-15000 ng / mL.

[0024] The volume ratio of the virus-like vector solution to the lysis solution is 1:2 to 1:9.

[0025] Preferably, when the viroid vector liquid adopts a crude viroid vector liquid, the volume ratio of the viroid vector liquid to the lysis liquid is 1:2-1:4.

[0026] Preferably, the virus-like vector liquid adopts a virus-like vector concentrate, and the volume ratio of the virus-like vector liquid to the lysis solution is 1:4 to 1:6.

[0027] Preferably, in step S1, the lysis conditions are: 25-37°C; 20-60min, standing for lysis.

[0028] Preferably, the pH value of the lysate is 7.4 to 8.0, more preferably 7.4.

[0029] The plasmid map of the pMD.2g is the well-known plasmid pMD2G in the Chinese patent publication number CN110684804A; The plasmid map of the pRSV-Rev is the pRSV-REV in the Chinese patent publication number CN110684804A; The main plasmid 1 is a plasmid expressing the long-chain protein of the vector GagPol, and the plasmid map is a plasmid expressing the long-chain protein of the lentivirus GagPol in the Chinese patent with publication number CN110684804A; The main plasmid 2 is a plasmid of the modified GagPol long-chain protein, and the plasmid map is a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein in the Chinese patent publication number CN110684804A; The main plasmid 3 is a plasmid for expressing the required Cas9 protein, and the plasmid map is a plasmid for expressing the protein required for forming RNP in the Chinese patent with publication number CN110684804A; The main plasmid 4 is a plasmid for expressing the desired sgRNA, and the plasmid map is a plasmid for expressing an exogenous target RNA containing an RNA stem-loop structure recognized by an RNA binding protein in the Chinese patent publication number CN117343153A.

[0030] The main plasmid 5 is a plasmid for expressing the desired sgRNA, and the plasmid map is a plasmid for expressing an exogenous target RNA containing an RNA stem-loop structure recognized by an RNA binding protein in the Chinese patent publication number CN117343152A.

[0031] Preferably, the preparation method comprises: growing 293T cells in the logarithmic growth phase at 2 to 4×10 5 Cells / cm 2 The cells were inoculated at a density of 100 μg / mL and cultured in DMED medium containing 2 to 10% FBS. After culturing in a 37°C incubator for 24 to 48 h, the plasmid, PEI and culture medium were mixed and added dropwise to the culture dish, which was then placed in a 37°C, 5% CO2 incubator for continued culture. The medium was changed after 4 to 6 hours, and an equal volume of fresh culture medium (DMEM containing 2 to 10% FBS) was added for continued culture. The crude vector venom was harvested 48 to 72 hours after transfection.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) The VLP-specific lysis solution developed in the present invention is more efficient than the traditional single-component lysis method (such as Triton X-100) and can achieve better lysis effect in a relatively short time; (2) Compared with conventional commercial lysis buffers (such as RIPA lysis buffer), the VLP-specific lysis buffer developed by the present invention has a higher concentration of Triton X-100, and an additional lysis component Tween 20 is added, as well as protease inhibitors to prevent protein degradation. This makes the lysis effect on VLP more efficient, and can more fully and conveniently lyse the vector at a low usage amount, in a shorter time, and at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 This is a flow chart of the method for detecting a virus-like vector of the present invention; Figure 2 The results of the test on the cleavage effect of RIPA on VLP1-CR in Example 1; Figure 3 The results of the test on the cleavage effect of each lysate on VLP1-UF in Example 2; Figure 4 The results of the lysate suitability test in Example 3 are as follows; Figure 5 The results of the lysate usage ratio test in Example 4 are as follows; Figure 6 This is a diagram of the structure of a virus-like vector. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0035] This protocol is only applicable to virus-like vectors that encapsulate Cas9 protein, such as Figure 6 .

[0036] The raw materials and equipment parameters involved in the following examples and comparative examples are as follows: The plasmid map of the pMD.2g is the well-known plasmid pMD2G in the Chinese patent publication number CN110684804A; The plasmid map of the pRSV-Rev is the pRSV-REV in the Chinese patent publication number CN110684804A; The main plasmid 1 is a plasmid expressing the long-chain protein of the vector GagPol, and the plasmid map is a plasmid expressing the long-chain protein of the lentivirus GagPol in the Chinese patent with publication number CN110684804A; The main plasmid 2 is a plasmid of the modified GagPol long-chain protein, and the plasmid map is a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein in the Chinese patent publication number CN110684804A; The main plasmid 3 is a plasmid for expressing the required Cas9 protein, and the plasmid map is a plasmid for expressing the protein required for forming RNP in the Chinese patent with publication number CN110684804A; The main plasmid 4 is a plasmid for expressing the desired sgRNA, and the plasmid map is a plasmid for expressing an exogenous target RNA containing an RNA stem-loop structure recognized by an RNA binding protein in the Chinese patent publication number CN117343153A.

[0037] The reagents are shown in Table 1: Table 1

[0038] The equipment is shown in Table 2: Table 2

[0039] Detection method: The Cas9 protein ELISA detection kit produced by Kaika Biotech was used to measure the Cas9 protein concentration in the carrier solution before and after lysis.

[0040] Operation procedure: Determine the Cas9 concentration according to the kit instructions.

[0041] The background value has been deducted from the test results to eliminate the influence.

[0042] Preparation Example 1 Carrier Liquid Preparation 1. VLP1 vector crude venom (VLP1-CR): Six plasmids, pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 4, were combined in a molar ratio of 1:1:1:1:2:2. 293T cells in the logarithmic growth phase were cultured at 4 × 10 5 Cells / cm 2 The cells were inoculated at a density of 100 μg / mL and cultured in DMED medium containing 10% FBS. After 48 h of culture in a 37 °C incubator, the plasmid, PEI and culture medium were mixed and added dropwise to the culture dish, which was then placed in a 37 °C, 5% CO2 incubator for continued culture. The medium was changed every 4 to 6 hours, and an equal volume of new culture medium (DMEM containing 10% FBS) was added for continued culture. The cells were harvested 72 hours after transfection to obtain the crude venom of the VLP1 vector.

[0043] 2. VLP2 vector crude venom (VLP2-CR): Six plasmids, pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main plasmid 5, were combined in a molar ratio of 1:1:1:1:2:2 and cultured in 293T cells in the logarithmic growth phase at 4×10 5 Cells / cm 2The cells were inoculated at a density of 100 μg / mL and cultured in DMED medium containing 10% FBS. After culturing in a 37°C incubator for 24 to 48 h, the plasmid, PEI and culture medium were mixed and added dropwise to the culture dish, which was then placed in a 37°C, 5% CO2 incubator for continued culture. The medium was changed after 4 to 6 hours, and an equal volume of new culture medium (DMEM containing 10% FBS) was added for continued culture. The cells were harvested 72 hours after transfection to obtain the crude venom of the VLP2 vector.

[0044] 3. Carrier Concentrate VLP1 vector concentrate (VLP1-UF): VLP1-CR is subjected to the above-mentioned centrifugal filtration, concentration and liquid exchange and chromatography purification operations to obtain VLP1-UF.

[0045] VLP2 vector concentrate (VLP2-UF): VLP2-CR is subjected to the above-mentioned centrifugal filtration, concentration and liquid exchange and chromatography purification operations to obtain VLP2-UF.

[0046] The steps for carrier concentration are as follows: (1) Centrifugal filtration: Although the cells used in the vector packaging system are adherent systems, cell death is inevitable during the culture process, so there are some impurities such as large cell fragments in the suspended vector solution. Although there is a deep filtration step in the subsequent process, in order to avoid the fragments and impurities clogging the filter membrane and increasing the pressure, thereby damaging the activity of the vector, we choose to centrifuge and deep filter the crude venom solution at a low speed to remove large impurities in the crude vector solution.

[0047] Centrifuge speed: 2500 g, Centrifugation temperature: 4 ℃, Centrifugation time: 5 ~ 7 min Deep filtration parameters: Membrane pore size: 0.45 μm, Filter membrane material: hydrophilic polyethersulfone, Use a peristaltic pump to slowly pump the crude carrier venom solution through the filter membrane and collect the filtrate.

[0048] (2) Concentrated solution replacement: The environment of the original carrier liquid may not be suitable for lysis experiments, so it is necessary to change the environment of the carrier to conditions suitable for lysis experiments, while removing some small molecule impurities in the carrier liquid, such as nucleic acid fragments, BSA, etc.

[0049] Ultrafiltration concentration parameters: Membrane pore size: 100 ~ 500 kDa, Membrane material: hydrophilic polyethersulfone, Buffer environment: 50 mM Tris-HCl buffer containing 20 mM EDTA, pH = 7.4 ~ 8.0.

[0050] Process flow: The filtrate after centrifugal filtration in step (1) is concentrated to one tenth of the original volume by ultrafiltration.

[0051] (3) Chromatographic purification: By utilizing the characteristics of various fillers, the carrier is separated from other impurities to achieve the purpose of purification.

[0052] Packing Type: Anion chromatography fillers, cationic chromatography fillers, affinity chromatography fillers, molecular sieve chromatography fillers or composite chromatography fillers, etc.

[0053] Chromatographic purification parameters: Due to the different types of fillers used, please refer to the corresponding instructions for use of the filler.

[0054] The composite chromatography filler Capto Core 700 was used in this preparation example.

[0055] p24 concentration: VLP1-CR 333.78 ng / mL; VLP1-UF 1505.35 ng / mL; VLP2-CR 287.36 ng / mL; VLP2-UF 1324.73 ng / mL.

[0056] Example 1 In this example, the commercial lysis buffer RIPA was first used to test the lysis effect on VLPs.

[0057] Lysis buffer (RIPA) recipe: 50 mM Tris (pH=7.4), 20 mM EDTA, 150 mM NaCl, 1 vol% TritonX-100, 0.1 vol% SDS, 1 wt% sodium deoxycholate.

[0058] The commercial lysis buffer RIPA was supplemented with 1 vol% protease inhibitor cocktail (the protease inhibitor cocktail contained 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64 and 1 mM Leupeptin, and the solvent was high-purity DMSO).

[0059] In order to prevent inappropriate lysis conditions from affecting the subsequent lysis solution formulation development, this experiment first conducted preliminary testing and evaluation of different lysis temperatures and lysis solution addition ratios. The lysis test experimental conditions were designed according to the instructions for use of the commercial RIPA lysis solution. Since the commercial RIPA lysis solution usually lyses solid samples (such as cells and tissues), there are no recommended lysis parameters for samples in the form of suspensions such as VLPs, and the addition of lysis solution will cause a certain dilution effect on the sample. Therefore, for the addition ratio of the lysis solution, we chose a higher ratio condition than that in the instructions, making it equal and excessive to the target sample, that is, sample volume: lysis solution volume = 1:1 and 1:2. For the lysis time, the 20 min recommended in the instructions was selected, and three commonly used lysis temperatures (low temperature 4°C, room temperature 25°C, and high temperature 37°C) were tested.

[0060] Operating parameters: The volume of VLP1-CR in Preparation Example 1: 50 μL, Lysis buffer volume: 50 μL (VLP1-CR and lysis buffer volume ratio 1:1), 100 μL (VLP1-CR and lysis buffer volume ratio 1:2), Cracking temperature: 4℃, 25℃, 37℃, Lysis time: 20 min, p24 concentration of VLP1-CR: 333.78 ng / mL.

[0061] Detection method: The Cas9 protein ELISA detection kit produced by Kaixia Biotechnology was used to measure the Cas9 protein concentration in the carrier solution before and after lysis.

[0062] Operation procedure: Determine the Cas9 concentration according to the kit instructions.

[0063] The process of testing the lysis effect of virus-like vectors is as follows Figure 1 shown.

[0064] Test results such as Figure 2 Therefore, judging from the results of the Cas9 protein concentration determination, when using RIPA lysis buffer to perform a lysis test on VLP1-CR, the effect is optimal when a 1:2 ratio is used as the lysis buffer addition ratio at 25°C. This condition can be used for subsequent lysis buffer formula development.

[0065] Example 2 Development of the lysate formulation of the present invention In this example, the components and concentrations of the lysate were optimized, and lysates 1-6 were designed, as shown in Table 3. The conditions of the preliminary test in Example 1 were used to develop a special lysate formula for VLP.

[0066] Operating parameters: The volume of the VLP1-UF carrier solution of Preparation Example 1: 50 μL, Lysis buffer volume: 100 μL, Cracking temperature: 25 ℃, Lysis time: 20 min, p24 concentration of VLP1-UF: 1505.35 ng / mL.

[0067] Detection method: The Cas9 protein ELISA detection kit produced by Kaixia Biotechnology was used to measure the Cas9 protein concentration in the carrier solution before and after lysis.

[0068] Operation procedure: Determine the Cas9 concentration according to the kit instructions.

[0069] Control group The lysis buffer formula of the control group 1 used in this example is a formula in which (1 vol% protease inhibitor) is added to the RIPA lysis buffer; the operating parameters are the same as those of lysis buffers 1 to 6.

[0070] Table 3 Lysis solution composition formula

[0071] Among them: 1 vol% protease inhibitor cocktail (protease inhibitor cocktail contains 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64 and 1 mM Leupeptin, the solvent is high-purity DMSO) The test results of the lysates 1-6 of the present invention and the control group (three parallel experiments) are as follows Figure 3 As shown: From the test results of Cas9 protein concentration, under the conditions of a volume ratio of carrier liquid to lysis liquid of 1:2, a lysis temperature of 25 °C, and a lysis time of 20 min, the lysis effects of lysis liquids 3, 5, and 6 were significantly better than those of the control group RIPA lysis liquid. However, after the experiment, it was found that detergent precipitation would occur in lysis liquid 5 after long-term storage, and it was irreversible and not conducive to storage. Therefore, lysis liquids 3 and 6 were selected for subsequent lysis condition research.

[0072] Example 3 Applicability test and formula confirmation of the lysis solution of the present invention 1. In order to ensure the universality of the lysis effect of the lysis solution of the present invention on VLP samples of different projects, this example carried out lysis tests on crude toxin and concentrated samples (VLP1-CR, VLP1-UF, VLP2-CR and VLP2-UF) of different project vectors.

[0073] Operating parameters: The volume of each VLP carrier solution in Preparation Example 1: 50 μL, Lysis buffer volume: 100 μL (1:2), Cracking temperature: 25 ℃, Lysis time: 20 min.

[0074] The p24 concentration of vector samples: VLP1-CR 333.78 ng / mL; VLP1-UF 1505.35 ng / mL; VLP2-CR 287.36 ng / mL; VLP2-UF 1324.73 ng / mL.

[0075] like Figure 4 As shown in the figure, lysis buffer 6 is also applicable to the lysis effect of crude VLP samples of different projects, and is significantly better than the control group. However, the lysis effect of concentrated samples did not meet expectations (combined with the concentration multiple relationship of each sample). It is expected that adjusting the ratio of lysis buffer used can solve this problem.

[0076] 2. Confirmation of the ratio parameters of the lysis buffer According to the results of step 1, the ratio of carrier solution to lysis solution = 1:2 is not enough to completely lyse the carrier at a high concentration. Therefore, this experiment finally confirmed the ratio of lysis solution.

[0077] Operating parameters: The volume of each VLP carrier solution in Preparation Example 1: 50 μL, Lysis buffer formula: Lysis buffer 6 Lysis buffer volume: 100 μL (1:2), 200 μL (1:4), 300 μL (1:6), 400 μL (1:8) Cracking temperature: 25 ℃, Lysis time: 20 min.

[0078] Vector sample p24 concentration: VLP2-CR 287.36 ng / mL; VLP2-UF 1324.73 ng / mL The experimental results are as follows Figure 5As shown: The results show that the carrier in the crude virus sample can be lysed when the lysis solution ratio is 1:2. For concentrated high-concentration samples, the ratio must be increased to at least 1:6. At this time, the Cas9 concentration and its corresponding crude virus sample concentration are consistent with the concentration multiple relationship. Continuing to increase the ratio to 1:8 will not make the lysis effect better. On the contrary, it may cause the sample to be diluted too much, causing the OD value in the ELISA test process to gradually tend to the detection limit, causing more experimental errors. Therefore, the ratio of lysis solution is determined to be carrier solution: lysis solution = 1:2 (applicable to crude virus samples) ~ 1:6 (applicable to concentrated samples).

[0079] In summary, the formula described in lysis buffer 6 can be used to complete the lysis operation of VLP samples of different projects at a lysis buffer ratio of 1:2 to 1:6, room temperature, and 20 minutes. The lysis effect is better than that of conventional commercial lysis buffer.

[0080] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A virus-like vector lysis solution, characterized in that: It is prepared from materials containing the following components and amounts: Tris 50-60 mM, EDTA 10-20 mM, NaCl150-200mM, TritonX-1001-3vol%, Sodium deoxycholate 0.5-1 wt%, SDS0.5- 0.1 vol%, Tween 201-2 vol% Protease inhibitor cocktail 1-2 vol%.

2. The virus-like vector lysis solution according to claim 1, characterized in that It is prepared from raw materials containing the following components and amounts: Tris 50 mM, EDTA 20 mM, NaCl 150 mM, TritonX-1002 vol%, Sodium deoxycholate 1 wt%, SDS0.1 vol%, Tween 202 vol%, Protease inhibitor cocktail 1 vol%.

3. The virus-like vector lysis solution according to claim 1, characterized in that The protease inhibitor cocktail contains 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64 and 1 mM Leupeptin, and the solvent is high-purity DMSO.

4. The virus-like vector lysis solution according to claim 1, characterized in that The Cas9 protein is encapsulated in the virus-like vector.

5. The virus-like vector lysis solution according to claim 1, characterized in that The method for obtaining the virus-like vector is to transfect a plasmid containing a gene sequence expressing the virus-like vector structure into a virus production cell to obtain the virus-like vector.

6. The virus-like vector lysis solution according to claim 5, characterized in that The plasmids containing the gene sequences for expressing the viral vector structure include pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3 and main plasmid 4.

7. The virus-like vector lysis solution according to claim 5, characterized in that The plasmids containing the gene sequences for expressing the viral vector structure include pMD.2g, pRSV-Rev, main plasmid 1, main plasmid 2, main plasmid 3, and main-main plasmid 5.

8. A method for lysing a virus-like vector, wherein the virus-like vector contains a Cas9 protein, characterized in that: The method comprises the following steps: S1. Using the lysis solution of any one of claims 1 to 7 to lyse the virus-like vector solution; S2. Detect the Cas9 protein content in the substances released after vector lysis.

9. The cracking method according to claim 8, characterized in that: The virus-like vector liquid includes a crude virus-like vector liquid, or a concentrated virus-like vector liquid obtained by centrifugal filtration, concentration and liquid replacement, and chromatography purification of the crude virus-like vector liquid.

10. The cracking method according to claim 9, characterized in that: When the virus-like vector liquid adopts a crude virus-like vector liquid, the volume ratio of the virus-like vector liquid to the lysis liquid is 1:2-1:4; The virus-like vector liquid adopts a virus-like vector concentrated liquid, and the volume ratio of the virus-like vector liquid to the lysis liquid is 1:4 to 1:6.

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