A universal method for rapid separation and purification of high-purity, high-quality small nuclei of Tetrahymena

Abstract

The present invention belongs to the field of biotechnology, and provides a method for rapid separation and purification of high-purity, high-quality small nuclei that is common to Tetrahymena. The following steps are included: 1) cell culture, treatment and collection; 2) cell disruption; 3) initial extraction of small nuclei; 4) deep purification of small nuclei. By utilizing the characteristics of small nuclei being separated from large nuclei during cell division, the separation of large and small nuclei without external artificial force is achieved; by using liquid nitrogen freezing, the separation of large and small nuclei is further assisted and the flexibility of experimental time is improved; by using a homogenizer, stable and gentle cell disruption is achieved; by using an improved centrifugation scheme, rapid purification of small nuclei with relatively high purity is achieved; by using a filter membrane to further remove large nuclei, purification of high-purity small nuclei is achieved. The method of the present invention is suitable for the separation and purification of small nuclei of various Tetrahymena, and has many advantages such as strong versatility, small nuclear damage, high stability, high separation purity, and short experimental cycle.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for rapidly separating and purifying high-purity, high-quality small nuclei common to Tetrahymena. The method is suitable for rapidly separating and purifying high-purity, high-quality small nuclei of Tetrahymena. Background Art

[0002] Most of the genetic information of eukaryotic organisms is stored in the cell nucleus. The information contained in the nucleus, such as the genome sequence, epigenetic modification, and chromatin three-dimensional structure, is the core of related research in modern genetics, evolutionary biology, cell biology, and developmental biology. Obtaining high-quality, synchronous / state cell nuclear samples is an important prerequisite for the success and high credibility of various biological experiments.

[0003] Tetrahymena is an important model organism, and its related research has made important contributions to the development of biology, such as the discovery of telomeres and telomerase (Nobel Prize in 2009) and the discovery of ribozymes (Nobel Prize in 1989). This species has dimorphism of the nucleus, that is, a cell has both a reproductive nucleus (micronucleus) responsible for inheritance and a nutritional nucleus (macronucleus) that ensures life activities. Usually, the micronucleus is embedded in the depression of the macronucleus, and separates from the macronucleus during the cell division period to start mitosis. The two nuclei have obvious differences in sequence, modification, and higher-order structure, providing a good research object for studies such as transcriptional regulation and DNA editing. It is of great biological significance to separate and purify the macronucleus and the micronucleus, and to carry out various experiments and compare them separately. However, since the number of chromosome copies of the macronucleus (about 90 fold) is much higher than that of the small nucleus (2 fold), at the chromosome level, a very small amount of macronucleus contamination will seriously affect the purity of the small nucleus chromosomes: in a 90% pure small nucleus sample, the proportion of small nucleus chromosomes is only 17%. Therefore, the purification of small nuclei has higher technical requirements and difficulty than the purification of large nuclei. There are more than ten Tetrahymena macronuclear genomes, but only one micronuclear genome. The lack of rapid and high-purity micronuclear sample technology has also restricted the development of related research.

[0004] The existing method for separating and purifying the small nucleus of Tetrahymena was developed for the model species Tetrahymenathermophila. Generally, the small nucleus is purified by differential centrifugation or density gradient centrifugation after cell disruption using a blender. This method is time-consuming, has poor stability, and the cell disruption process is very intense, which not only damages the small nuclear DNA, but also produces large nuclear fragments that are difficult to distinguish from the small nucleus. More importantly, the previous conventional methods have poor versatility and can only be used to separate and purify the small nucleus of Tetrahymena thermophila. When used for the extraction of small nuclei of other Tetrahymena, blender disruption cannot separate the small nucleus from the large nuclear depression, and differential centrifugation and density gradient centrifugation schemes cannot effectively distinguish between large and small nuclei, resulting in failure of separation and purification of small nuclei.

[0005] Therefore, in order to solve the above problems, the applicant has developed a method for rapid separation and purification of high-purity, high-quality small nuclei that is universal for Tetrahymena. Summary of the invention

[0006] The purpose of the present invention is to provide a high-purity, high-quality small nucleus rapid separation and purification method that is universal for Tetrahymena, and achieves the following technical purposes: 1) utilizing the characteristic that small nuclei are separated from large nuclei during cell division to achieve separation of large and small nuclei without external artificial force; 2) utilizing liquid nitrogen freezing to further assist in the separation of large and small nuclei and improve the flexibility of experimental time; 3) utilizing a homogenizer to achieve stable and gentle cell disruption; 4) utilizing an improved centrifugation scheme to achieve rapid purification of relatively high-purity small nuclei; 5) utilizing a filter membrane to further remove large nuclei to achieve purification of high-purity small nuclei.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a method for rapid separation and purification of high-purity, high-quality small nuclei of Tetrahymena, comprising the following steps:

[0008] 1) Cell culture, treatment and collection: After culturing Tetrahymena cells in SPP medium, the cells were collected by centrifugation, washed twice with Tris buffer, and resuspended in Tris buffer (10 mM, pH 7.4) for starvation treatment. The starved cells were collected by centrifugation, resuspended in SPP medium and cultured for 4 to 5 hours, washed and collected with Tris buffer (10 mM, pH 7.4), and the collected cells were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use;

[0009] 2) Cell disruption: Take the frozen cells in step 1) at 4°C or on ice, thaw and mix with Medium A solution to disrupt the cells;

[0010] 3) Initial extraction of small nuclei: Centrifuge the disrupted cells in step 2) for 3 minutes, retain the precipitate, transfer the supernatant and mix well, then centrifuge again, repeat the operation 4-6 times; the precipitate collected in the first 1-2 centrifuges is mainly large nuclei, and the precipitate collected in subsequent centrifuges is crudely extracted high-purity small nuclei;

[0011] 4) Deep purification of small nuclei: Filter the crude high-purity small nuclei obtained in step 3), resuspend them in Medium A solution, and then filter them preliminarily with a cell sieve to remove impurities in the sample. Transfer and centrifuge for 3 minutes, repeat several times, and then filter them again to obtain small nuclei with a purity of 100%.

[0012] Wherein, the SPP medium described in step 1) comprises the following components (mass percentage): 1% Proteosepeptone, 0.1% Yeast extract, 0.2% Glucose, 0.003% Sequestrene;

[0013] The SPP culture medium preparation method is as follows: dissolving the components in water according to proportion, and sterilizing at 121° C. for 15 minutes.

[0014] The Tris buffer used in all steps is the same, and the preparation method is to dissolve Tris-HCl powder in water to a final concentration of 10 mM and adjust the pH to 7.4.

[0015] Preferably, in step 1), the Tetrahymena cells are cultured until the cell concentration in the culture medium is 4 to 5*10 6 cells / mL, centrifuged at 1500r / min for 3 minutes to collect Tetrahymena cells, washed twice with Tris buffer (10mM, pH 7.4), and resuspended in Tris buffer, starved for 12 to 24 hours, collected the starved cells by centrifugation, resuspended in SPP medium, 4 to 5 hours later, washed with Tris buffer (10mM, pH 7.4) and collected into 2mL EP tubes, the collected cells were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

[0016] Wherein, the Medium A solution described in steps 2) and 4) is prepared and used immediately (Note: adding it to the pre-prepared Medium A solution in advance will cause problems such as solution instability, volatilization, and inactivation), and 100 μL 0.1 M PMSF, 50 μL n-octanol, 20 μL 0.5 M EDTA and 20 μL NP-40 are added to each 9 mL of the pre-prepared Medium A solution to obtain a Medium A solution; the pre-prepared Medium A solution comprises the following components (% refers to mass percentage): 3% Sucrose, 4% Gum arabic, 5mM PIPES, 5mMMgCl2, 5mM Spermidine and 1mM EDTA, pH 6.75.

[0017] The method for preparing the Medium A prefabricated solution is as follows: dissolving the components in water according to proportion, and sterilizing at 121° C. for 15 minutes.

[0018] Preferably, in step 2), the cells are disrupted by homogenizing the cells up and down 50 to 60 times using a glass homogenizer.

[0019] Preferably, in step 3), the broken cells are transferred to 1100-1200g centrifugation for 3 minutes, the precipitate is retained, and the upper layer and the supernatant are transferred and mixed and then centrifuged again, and the operation is repeated 4-6 times; usually, the precipitate collected in the first 1-2 centrifugations is mainly large nuclei, and the precipitate collected thereafter is small nuclei with a purity of >95%. In this step, the precipitate collected in the first 1-2 centrifugations is mainly large nuclei, and the precipitates collected thereafter are small nuclei with a purity of >95%;

[0020] Preferably, in step 4), the small nuclei with a purity of >95% obtained in step 3) are filtered: the small nuclei sample is resuspended in 1 mL of Medium A solution, and then preliminarily filtered with a 5 μm cell sieve to remove impurities in the sample, transferred to an ultrafiltration centrifuge tube, and centrifuged at 550 r / min for 3 minutes. After filtration, small nuclei with a purity of 100% are obtained.

[0021] Among them, the purity in steps 3) and 4) is calculated as follows: take the sample to be tested, a single sample volume of 10 μL, place it under a microscope, count the total number of cell nuclei, and when the total number of cell nuclei is greater than 100, calculate the proportion of small nuclei in the sample (recorded number of small nuclei / recorded total number of cell nuclei), take samples 5 to 10 times, and take the average value, which is the small nucleus purity.

[0022] In the technical scheme of a high-purity, high-quality small nucleus rapid separation and purification method that is universal for Tetrahymena provided by the present invention, the key steps and principles are: 1. By supplementing nutrients to the starved cells, the cells are caused to divide synchronously to achieve damage-free separation of large and small nuclei. 2. Liquid nitrogen is used to quickly freeze and fix the cells to further assist in the separation of large and small nuclei. 3. A homogenizer is used instead of a blender to achieve gentler cell disruption. 4. Small nucleus samples with a purity of >95% are quickly obtained through an innovative centrifugation scheme (1100-1200g, 3 minutes). 5. The large nuclei are further removed by filtration using a membrane to obtain a small nucleus sample with a purity of 100%.

[0023] The key components in the technical solution provided by the present invention are: Medium A solution; the key role played in achieving the technical effect is: 3% Sucrose and 4% Gum arabic provide a suitable density for the solvent; 5mM PIPES provides a pH buffer system for the solvent; 5mM MgCl2 and 5mM Spermidine have a protective effect on DNA; EDTA maintains the cell nucleus at a suitable physical strength; PMSF inhibits the activity of digestive enzymes in the cell; n-octanol provides an upward pulling force for cell components, and NP-40 helps the cell nucleus migrate in the solvent.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] 1. A simple and universal method for non-destructive separation of large and small nuclei of Tetrahymena is proposed. The synchronization of Tetrahymena cells is achieved by starvation treatment, so that they divide synchronously after nutrition is supplemented. The separation of large and small nuclei without external human forces is achieved by taking advantage of the characteristic of the biological group itself, that is, "the small nucleus separates from the large nucleus in the early stage of Tetrahymena division." Taking Tetrahymena pigmentosa as an example, the separation ratio of large and small nuclei obtained by the previous method is often less than 50%; while with this method, the separation ratio is higher than 90%.

[0026] 2. The cells were fixed by quick freezing in liquid nitrogen, which enabled flexibility in experimental time. At the same time, this treatment helped to destroy the connection between the large and small nuclei without causing DNA to be degraded by intracellular enzymes.

[0027] 3. Using a homogenizer instead of a blender achieves gentler cell disruption. It reduces large nuclear fragments generated by mechanical force and prevents damage to small nuclear DNA. At the same time, compared with a blender, using a homogenizer for disruption has higher stability and can reduce batch-to-batch differences. In addition, compared with a blender, using a homogenizer for disruption can reduce the volatilization of toxic reagents during the experiment and better protect the experimenters.

[0028] 4. A centrifugation protocol for quickly obtaining small nucleus samples with a purity of >95% for Tetrahymena is proposed. This protocol greatly shortens the time required. Compared with the previous protocol that took up to 4 hours, this protocol can be completed within 30 minutes, greatly reducing the risk of DNA degradation due to excessive experimental time. More importantly, this protocol has a wider applicability. Taking Tetrahymena pigmentosa as an example, the previous conventional protocol can often only increase the purity of small nuclei to 80%, while this protocol can quickly obtain small nucleus samples with a purity of >95%.

[0029] 5. Use the filter membrane to further filter and remove the large nuclei to obtain a small nucleus sample with a purity of 100%. This step plays an important role in increasing the proportion of small nucleus chromosomes at the chromosome level. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The data in this specification only represent some embodiments of the present invention, rather than all embodiments. It should be understood that the data for specific species shown in this specification are only for the purpose of illustrating the present invention, and are not intended to limit the present invention. For professionals in this field, without paying creative labor, high-purity micronucleus samples of other Tetrahymena can still be obtained by separation and purification according to this method.

[0031] Meanwhile, throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0032] Figure 1 The experimental flow chart of the present invention and the comparison with the previous method;

[0033] Figure 2 The effect of cell disruption and micronucleus separation and purification using this method is demonstrated by taking Tetrahymena pigmentosa as an example;

[0034] Figure 3 The effect of small nuclei purification by this method is demonstrated by taking T. thermophila and Tetrahymena borealis as examples;

[0035] Figure 4 The effect of conventional cell disruption and micronucleus separation and purification is demonstrated using Tetrahymena pigmentosa as an example.

[0036] Figure 5 The effect of cell disruption and micronucleus separation and purification without starvation treatment is demonstrated using Tetrahymena pigmentosa as an example.

[0037] Figure 6 The small nucleus purification effect after changing the small nucleus extraction centrifugation protocol is demonstrated using Tetrahymena pigmentosa as an example. DETAILED DESCRIPTION

[0038] Hereinafter, the applicant will describe the present invention in more detail through examples. The following examples are only used to specifically illustrate the implementation method of the present invention, but they should not be understood as limiting the scope of protection of the present invention.

[0039] The names, ingredients and preparation methods of the reagents used in the following examples are as follows:

[0040] SPP medium includes the following components (mass percentage): 1% Proteose peptone, 0.1% Yeast extract, 0.2% Glucose, 0.003% Sequestrene;

[0041] The SPP culture medium preparation method is as follows: dissolving the above components in water according to proportion, and sterilizing at 121° C. for 15 minutes.

[0042] The Tris buffer was prepared by dissolving Tris-HCl powder in water to a final concentration of 10 mM and adjusting the pH to 7.4.

[0043] Medium A pre-made solution includes the following components (% refers to mass percentage): 3% Sucrose, 4% Gumarabic, 5mM PIPES, 5mM MgCl2, 5mM Spermidine and 1mM EDTA, pH 6.75;

[0044] The method for preparing the Medium A prefabricated solution is as follows: dissolving the above components in water according to proportion, and sterilizing at 121° C. for 15 minutes.

[0045] Medium A solution was prepared and used immediately (Note: adding to the pre-made Medium A solution in advance will cause problems such as solution instability, volatilization, and inactivation). To each 9 mL of the pre-made Medium A solution, 100 μL of 0.1 M PMSF, 50 μL of n-octanol, 20 μL of 0.5 M EDTA, and 20 μL of NP-40 were added to obtain the Medium A solution;

[0046] The calculation method of small nuclear purity in the following embodiments is as follows: take the sample to be tested, a single sampling volume of 10 μL, place it under a microscope, count the total number of cell nuclei, and when the total number of cell nuclei is greater than 100, calculate the proportion of small nuclei in the sample (recorded number of small nuclei / recorded total number of cell nuclei), take samples 5 to 10 times, and take the average value, which is the small nuclear purity.

[0047] Example 1

[0048] Taking T. pigmentosa, T. thermophila and T. borealis as examples, three groups were divided to complete the following culture steps, and the culture objects were T. pigmentosa cells, T. thermophila cells and T. borealis cells respectively:

[0049] 1) Cell culture, processing and collection

[0050] Tetrahymena cells were cultured in 100 mL SPP medium (1% Proteose peptone, 0.1% Yeast extract, 0.2% Glucose, 0.003% Sequestrene) until 4.5*10 6cells / mL. Collect Tetrahymena cells by centrifugation at 1500r / min for 3 minutes, wash twice with Tris buffer (10mM, pH7.4), and resuspend in 100mL Tris buffer (10mM, pH 7.4) for starvation for 16 hours. Collect the starved cells by centrifugation and resuspend in 100mL SPP medium. After 4 hours, wash with Tris buffer (10mM, pH 7.4) and collect the cells into 2mL EP tubes. Quickly freeze the collected cells in liquid nitrogen and store them in a -80℃ refrigerator for later use.

[0051] 2) Cell disruption

[0052] All the following steps were performed at 4°C or on ice. Take the frozen cells, thaw and mix in 9 mL of Medium A solution. Use a glass homogenizer to break the cells up and down 55 times.

[0053] 3) Initial small nucleus

[0054] The broken cells were transferred to a 2 mL EP tube and centrifuged at 1127 g for 3 minutes. The precipitate was retained and the supernatant and the supernatant were transferred to a new 2 mL EP tube. After mixing, the supernatant was centrifuged again. The operation was repeated 5 times to finally obtain small nuclei with a purity of 97%.

[0055] 4) Deep purification of small nuclei

[0056] The 97% small nuclei obtained in the previous step were filtered to increase the purity to 100%. Use 1mL Medium A solution to resuspend the small nuclei sample. Use a 5um cell sieve (pluriStrainer, Germany) for preliminary filtration to remove impurities in the sample. Transfer to a 5μm ultrafiltration tube (Ultrafree-CL Centrifugal Devices, Germany) and centrifuge at 550r / min for 3 minutes. The filtered sample is a small nucleus with a purity of 100%.

[0057] The cell disruption and micronucleus extraction results of Tetrahymena pigmentosa in this example are as follows: Figure 2 The implementation method described can be directly applied to other species of Tetrahymena without any changes. Figure 3 The results are shown for the purification of small nuclei from T. thermophila and T. borealis cells in this example.

[0058] Example 2: Separation and purification scheme with changing the duration of nutritional supplementation

[0059] The culture object is Tetrahymena pigmentosa, and the other steps are the same as those in Example 1, except that the culture time after resuspending in 100 mL of SPP culture medium in step 1) is 3 hours.

[0060] As a result, small nuclei with 100% purity can still be obtained. However, due to the short time of supplementation, by counting the proportion of small nuclei separated from large nuclei, it can be seen that about 1 / 3 of the small nuclei did not separate from the large nuclei, resulting in these small nuclei being lost in the subsequent centrifugation process.

[0061] Example 3: Separation and purification scheme with altered centrifugation time in step 3)

[0062] The culture object is Tetrahymena pigmentosa, and the other steps are the same as those in Example 1, except that the centrifugation condition in step 3) is: centrifugation at 1127 g for 5 minutes.

[0063] As a result, small nuclei with a purity of 100% can still be obtained. However, due to the long centrifugation time, more small nuclei were lost in the early centrifugation, resulting in the number of small nuclei collected being slightly lower than that in Example 1. According to the results of direct observation under a microscope, the number of small nuclei obtained in Example 3 is about 1 / 2 of that in Example 1.

[0064] Example 4: Separation and purification scheme with altered centrifugation time in step 4)

[0065] The culture object was Tetrahymena pigmentosa, and the other steps were the same as in Example 1, except that the centrifugation condition in step 4) was: 1000 r / min for 5 minutes. As a result, small nuclei of high purity were still obtained. However, due to the excessive centrifugal force, a small number of large nuclei were pressed into fragments and passed through the microfiltration centrifuge tube, causing a certain degree of contamination to the sample, and the purity of the small nuclei was about 98%.

[0066] Comparative Example 1 Conventional method

[0067] In the following, the process and effect of purifying micronuclei of Tetrahymena pigmentosa by conventional culture and purification method will be demonstrated. The SPP culture medium and Medium A solution used are the same as those in Example 1.

[0068] 1) Cell culture and collection. Cultivate T. pigmentosa cells in 1L SPP medium until 4.5*10 6 cells / mL and centrifuged at 1500r / min for 3 minutes to collect Tetrahymena cells.

[0069] 2) Cell disruption. Resuspend the collected cells in 200 mL of Medium A solution. Add 2 mL of 0.1 M PMSF and 1 mL of n-octanol. Use a blender to disrupt the cells at low speed for 10 seconds, then add 9 mL of 0.5 M EDTA and disrupt at high speed for another 30 seconds.

[0070] 3) Extraction of small nuclei. Transfer the broken cells to a 50mL centrifuge tube and centrifuge at 1000g for 10 minutes. Keep the precipitate and transfer the upper material and supernatant to a new 50mL centrifuge tube. Mix well and centrifuge again. Repeat the operation until almost no precipitate can be obtained. Increase the centrifugal force to 1100g and repeat the centrifugation. Increase the centrifugal force to 1300g and repeat the centrifugation. Increase the centrifugal force to 1400g and repeat the centrifugation. Increase the centrifugal force to 1500g and repeat the centrifugation. Increase the centrifugal force to 1600g and repeat the centrifugation. Increase the centrifugal force to 3000g and repeat the centrifugation. The purity of small nuclei obtained using conventional methods is only ~80% at most. Even without considering the contamination of large nuclear fragments, at the chromosome level, the proportion of small nuclear chromosomes is still <10%.

[0071] The extraction effect of the small nucleus in this comparative example is as follows Figure 4 shown. Figure 2 A and Figure 4 A comparison shows that, compared with the conventional method, the separation of large and small nuclei in the present invention is more gentle (large nuclei are more regular, and there are almost no large nuclei fragments) and sufficient (there are almost no unseparated large and small nuclei). Figure 2 F and Figure 4 B shows that the present invention has higher extraction efficiency and better effect than the conventional method.

[0072] Comparative Example 2 Method for separating large and small nuclei of Tetrahymena without treatment after cultivation

[0073] In the following, the process and effect of purifying small nuclei of Tetrahymena pigmentosa (T. pigmentosa) by directly collecting after culture without starvation, nutrition supplementation, and liquid nitrogen quick freezing treatment will be demonstrated. The remaining steps are the same as Example 1.

[0074] The SPP culture medium and Medium A solution used were the same as those in Example 1.

[0075] 1) Cell culture and collection. Cultivate T. pigmentosa cells in 100 mL SPP medium until 4.5*10 6 cells / mL, centrifuged at 1500r / min for 3 minutes to collect Tetrahymena cells. The collected cells were resuspended in 10mL Medium A solution.

[0076] 2) cell disruption, 3) initial extraction of small nuclei, and 4) deep purification of small nuclei are all the same as in Example 1.

[0077] The cell disruption and extraction effects of this comparative example are as follows: Figure 5 shown. Figure 2 A and Figure 5 As can be seen from the comparison in A, without starvation treatment, a large number of small nuclei are still embedded in the large nuclei after the cells are broken. Figure 2 E and Figure 5 Comparison with B shows that since the small nuclei are not separated from the large nuclei, a large number of small nuclei are lost during the extraction process, resulting in very few small nuclei being obtained in the end.

[0078] Comparative Example 3: Separation method after changing the centrifugation scheme for extracting small nuclei in step 3

[0079] In the following, the process and effect of purifying small nuclei of Tetrahymena pigmentosa (T. pigmentosa) by using the Tetrahymena large and small nuclei centrifugation method after changing the small nuclei extraction centrifugation scheme will be demonstrated, and the remaining steps are the same as in Example 1.

[0080] The SPP culture medium and Medium A solution used were the same as those in Example 1.

[0081] 1) Cell culture and collection, 2) Cell disruption, and 4) Deep purification of small nuclei are the same as in Example 1.

[0082] 3) Small nuclei extraction was performed according to the previous routine method. The broken cells were transferred to a 50mL centrifuge tube and centrifuged at 1000g for 10 minutes. The precipitate was retained, and the upper layer and supernatant were transferred to a new 50mL centrifuge tube. After mixing, the supernatant was centrifuged again and repeated until almost no precipitate could be obtained. The centrifugal force was increased to 1100g and the centrifugation was repeated. The centrifugal force was increased to 1300g and the centrifugation was repeated. The centrifugal force was increased to 1400g and the centrifugation was repeated. The centrifugal force was increased to 1500g and the centrifugation was repeated. The centrifugal force was increased to 1600g and the centrifugation was repeated. The centrifugal force was increased to 3000g and the centrifugation was repeated. The purity of small nuclei obtained using the previous method was only 83% at most. Even without considering the contamination of large nuclear fragments, the proportion of small nuclear chromosomes at the chromosome level is still <10%. The purity of small nuclei after filtration is only 92%. Even without considering the contamination of large nuclear fragments, the proportion of small nuclear chromosomes at the chromosome level is only 20%.

[0083] The extraction effect of the small nucleus in this comparative example is as follows Figure 6 shown. Figure 2 F and Figure 6 By comparison, it can be seen that the extraction efficiency and effect of the centrifugal solution of the present invention are higher.

Claims

1. A method for rapid separation and purification of the micronucleus of Tetrahymena, characterized in that: The steps include: 1) Cell culture, processing and collection: Tetrahymena cells were cultured in SPP medium and then centrifuged to collect (4-5)*10 6 cells / mL, centrifuged at 1500r / min for 3 minutes to collect Tetrahymena cells; resuspended in Tris buffer, starved for 12 to 24 hours, centrifuged to collect the starved cells, resuspended in SPP medium for 4 to 5 hours, washed and collected with Tris buffer, and the collected cells were quickly frozen in liquid nitrogen for later use; 2) Cell disruption: Take the frozen cells from step 1) at 4°C or on ice, thaw and mix with Medium A solution, and use a glass homogenizer to break the cells up and down 55 times; 3) Initial extraction of small nuclei: transfer the cells disrupted in step 2) to centrifuge at 1100-1200 g for 3 minutes, retain the precipitate, transfer the supernatant and mix well, and centrifuge again. Repeat the operation 4-6 times. The precipitate collected is the small nuclei with a purity of >95%; 4) Deep purification of small nuclei: Filter the small nuclei with a purity of >95% obtained in step 3): resuspend the small nuclei sample with Medium A solution, and then preliminarily filter with a 5 μm cell sieve to remove impurities in the sample, transfer to a 5 μm ultrafiltration centrifuge tube, centrifuge at 550 r / min for 3 minutes, and obtain small nuclei with a purity of 100% after filtration; In the steps 2) and 4), the Medium A solution is prepared and used immediately. To each 9 mL of the Medium A pre-prepared solution, 100 μL of 0.1 M PMSF, 50 μL of n-octanol, 20 μL of 0.5 M EDTA and 20 μL of NP-40 are added to obtain the Medium A solution; the Medium A pre-prepared solution comprises the following components: 3% Sucrose, 4% Gum arabic, 5mM PIPES, 5mM MgCl2, 5mM Permidine and 1mM EDTA, pH 6.

75.

2. A method for rapid separation and purification of the small nucleus of Tetrahymena according to claim 1, characterized in that: The SPP culture medium described in step 1) includes the following components in percentage by mass: 1% Proteose peptone, 0.1% Yeast extract, 0.2% Glucose, and 0.003% Sequestrene.

3. A method for rapid separation and purification of the small nucleus of Tetrahymena according to claim 1, characterized in that: The Tris buffer used in each step was 10 mM, pH 7.

4.

4. A method for rapid separation and purification of the small nucleus of Tetrahymena according to claim 1, characterized in that: The calculation method of small nucleus purity in steps 3) and 4) is as follows: take the sample to be tested, a single sample volume of 10 μL, place it under a microscope, count the total number of cell nuclei, and when the total number of cell nuclei is greater than 100, calculate the proportion of small nuclei in the sample, the recorded number of small nuclei / the recorded total number of cell nuclei, take samples 5 to 10 times, and take the average value, which is the small nucleus purity.

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