Research method for influence of chicken EZH2 gene on neoplastic diseases
阅读说明：本技术 一种鸡ezh2基因对肿瘤性疾病影响的研究方法 (Research method for influence of chicken EZH2 gene on neoplastic diseases ) 是由 郁川 朱文文 张贺伟 程相朝 田文静 宋敏杰 王聪慧 于 2021-06-29 设计创作，主要内容包括：本发明公开了一种鸡EZH2基因对肿瘤性疾病影响的研究方法,包括S1.纯化鸡EZH2原核蛋白,得到EZH2蛋白样品；S2.将纯化后的鸡EZH2原核表达蛋白与MSB1细胞共同培养,测定EZH2原核表达蛋白对MSB1细胞增殖的影响；S3.于不同时间提取细胞总RNA,检测MDV相关基因的表达水平并进行分析,测定EZH2原核表达蛋白对MDV相关基因的表达水平的影响；实验证明,鸡EZH2蛋白可影响MSB1细胞的增殖,且影响该细胞中MDV Meq和gB基因的表达,为进一步探究鸡EZH2在MDV感染致肿瘤中的作用机制研究奠定了基础。(The invention discloses a method for researching influence of chicken EZH2 gene on neoplastic diseases, comprising S1, purifying chicken EZH2 prokaryotic protein to obtain an EZH2 protein sample; s2, co-culturing the purified chicken EZH2 prokaryotic expression protein and an MSB1 cell, and determining the influence of the EZH2 prokaryotic expression protein on the MSB1 cell proliferation; s3, extracting total RNA of cells at different time, detecting and analyzing the expression level of the MDV related gene, and determining the influence of the EZH2 prokaryotic expression protein on the expression level of the MDV related gene; experiments prove that the chicken EZH2 protein can influence the proliferation of MSB1 cells and the expression of MDV Meq and gB genes in the cells, and lays a foundation for further researching the action mechanism of the chicken EZH2 in MDV infection-induced tumors.)
1. A method for researching the influence of chicken EZH2 gene on neoplastic diseases is characterized in that: comprises the steps of
S1, purifying chicken EZH2 prokaryotic protein to obtain a purified His tag EZH2 protein sample;
s2, co-culturing the purified chicken EZH2 prokaryotic expression protein and an MSB1 cell, and determining the influence of the EZH2 prokaryotic expression protein on the MSB1 cell proliferation;
s3, on the basis of the step S2, extracting total RNA of cells at different time points, detecting and analyzing the expression levels of the MDV related genes Meq and gB, and determining the influence of the EZH2 prokaryotic expression protein on the expression levels of the MDV related genes Meq and gB.
2. The method of claim 1, wherein the effect of chicken EZH2 gene on neoplastic disease is characterized by: the process for purifying the chicken EZH2 prokaryotic protein described in the step S1 comprises the following steps:
s101, performing induction culture on escherichia coli Rosetta containing a prokaryotic expression vector of chicken EZH2 for 8 hours at 29 ℃ by using 1mmol of an inducer IPTG, collecting and weighing bacterial precipitates, and adding non-denaturing lysis solution according to the proportion that 4ml of non-denaturing lysis solution is added into each gram of bacterial precipitates;
s102, adding lysozyme until the final concentration is 1mg/ml, then fully and uniformly mixing, and carrying out ice-water bath for 30 min;
s103, ultrasonically cracking bacteria on ice, centrifuging for 30min at 4 ℃ under 10000g, and collecting the centrifuged supernatant in an EP tube and placing the tube on an ice water bath or ice;
s104, taking 1ml of uniformly mixed 50% BeyogoldTMHis-tag Purification Resin, centrifuging at 4 ℃ and discarding the storage solution, adding 0.5ml of non-denatured lysate into the gel in the Purification column, centrifuging at 4 ℃ and discarding the equilibrium solution, repeating the equilibrium for 2-3 times and then adding the supernatant of the bacterial lysate;
s105, collecting the flow-through liquid and repeatedly loading the flow-through liquid on the column for 3-5 times;
s106, then, taking down the cover at the bottom of the purification column to enable liquid in the column to naturally flow out due to the influence of gravity;
s107, adding 0.5ml of non-denaturing washing liquid each time, and then continuously washing the column for 5-6 times, wherein 20ul of washing liquid is collected each time;
s108, eluting the target protein for 6-10 times by using 0.5ml of non-denatured eluent each time to obtain a purified His label EZH2 protein sample.
3. The method of claim 1, wherein the effect of chicken EZH2 gene on neoplastic disease is characterized by: the specific application process of co-culturing the chicken EZH2 prokaryotic expression protein and the MSB1 cell in the step S2 comprises the following steps:
s201, diluting the centrifugally collected cells to 5.0 x 10 by using 1640 growth solution5Taking 100 mu L/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic proteins with different concentrations into each well of a treatment group to enable the final concentrations to be respectively 10, 20, 40 and 50 mu g/mL, adding pet-32a empty carrier protein with the equivalent volume and the corresponding concentration into a control group, setting 4 times for each concentration, setting a normal cell culture control without an antibody, and slightly shaking and uniformly mixing;
s202, after culturing for 24 hours and 48 hours in a 37 ℃ cell culture box, adding 10 mu l of CCK-8 reagent into each hole, after continuously culturing for 4 hours, adjusting the blank control to zero, and measuring OD on an enzyme labeling instrument450。
4. The method of claim 1, wherein the effect of chicken EZH2 gene on neoplastic disease is characterized by: the specific process of applying the chicken EZH2 prokaryotic expression protein in the MDV related gene in the step S3 comprises the following steps:
s301, diluting the centrifugally collected cells to 1.0X 10 by using 1640 growth solution6Taking 1 mL/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic protein with different concentrations into each well of a treatment group to enable the final concentration to be 50 mu g/mL, adding pet-32a empty carrier protein with the corresponding concentration in the same volume into a control group, and setting each concentration to be 4 times;
s302, continuously culturing for 24h and 48h, collecting cells, and extracting MSB1 cell total RNA;
s303, detecting the MDV related gene expression by using a real-time fluorescent quantitative PCR method.
5. The method of claim 3 for studying the effect of chicken EZH2 gene on neoplastic diseases, wherein the method comprises the following steps: the MSB1 cell total RNA extraction process described in step S303 comprises
(1) Collecting 2X 106-1×107MSB1 cell. Centrifuging at 2000 Xg for 5min, and discarding the supernatant;
(2) adding 400 μ l buffer solution R-I, repeatedly sucking with syringe equipped with 21-25 gauge needle for 8-10 times, and transferring into 1.5ml centrifuge tube;
(3) add 150. mu.l buffer R-II and vortex for 1min, centrifuge at 12000 Xg for 5min at 4 ℃;
(4) transferring the supernatant to a 1.5ml microcentrifuge tube, adding 250. mu.l of isopropanol and mixing thoroughly;
(5) placing the preparative column in a 2ml microcentrifuge tube, transferring the binding mixture from step (4) to the preparative tube, and centrifuging at 6000 Xg for 1 minute;
(6) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, adding 700. mu.l of BufferW1A into the preparation tube, and centrifuging for 1 minute at 12000 Xg;
(7) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, adding 700. mu.l Buffer W2, centrifuging at 12000 Xg for 1min, and washing once with 700. mu.l Buffer W2 by the same method;
(8) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, and centrifuging for 1 minute at 12000 Xg;
(9) placing the prepared tube into a clean 1.5ml centrifuge tube, adding 70-100 μ l Buffer TE or RNase-free water into the center of the prepared tube membrane, standing at room temperature for 1min, centrifuging at 12000 Xg for 1min, and eluting to obtain RNA.
6. The method of claim 3 for studying the effect of chicken EZH2 gene on neoplastic diseases, wherein the method comprises the following steps: the process of detecting MDV related gene expression by the real-time fluorescent quantitative PCR method in step S304 comprises
(1) Removing the genome DNA reaction;
(2) carrying out reverse transcription reaction of MSB1 cell total RNA;
(3) preparing Real Time reaction liquid by using an operation method of performing Real Time PCR reaction by using a TB Green Premix Ex Taq II method;
(4) performing Real Time PCR reaction;
(5) and calculating the expression level of the MDV related gene, and performing data analysis.
7. The method of claim 6, wherein the effect of chicken EZH2 gene on neoplastic disease is characterized by: in the Real Time PCR reaction process of the step (3), a specific primer and a chicken internal reference gene are required to be added into the reaction liquid of the Real Time PCR, and the specific primer and the chicken internal reference gene comprise
MDV Meq：F 5'-GTCCCCCCTCGATCTTTCTC-3′
R 5'-CGTCTGCTTCCTGCGTCTTC-3′；
gB：F 5'-ACCCCATTCGGTGGCTTTTC-3′
R 5'-GCGTCCAGTTGTCTGAGG-3′；
GAPDH：F 5'-TCTATCTCTTTGCTGGGACT-3′
R 5'-CTACAGCCAACTTTCATCAG-3′。
8. The method of claim 1, wherein the effect of chicken EZH2 gene on neoplastic disease is characterized by: the chicken EZH2 protein can inhibit the proliferation of MSB1 cells and influence the expression of MDV Meq and gB genes in the MSB1 cells.
9. The method of claim 8 for studying the effect of chicken EZH2 gene on neoplastic diseases, wherein the method comprises the following steps: the chicken EZH2 protein has activity and can be applied to the research of the action mechanism of the chicken EZH2 protein in chicken tumor diseases.
Technical Field
The invention relates to the technical field of gene therapy, in particular to a method for researching influence of chicken EZH2 gene on tumor diseases.
Background
EZH2(enhancer of Zeste homolog 2), namely an enhancer of Zeste homolog 2, is a main component of PRC2 (polycombin inhibitory complex 2) and can also be called histone lysine methyltransferase, and researches and experiments in the field of epigenetics prove that targeted EZH2 inhibitors can inhibit the carcinogenic activity of PRC2 and can form certain barrier effect on the occurrence, invasion and diffusion of cancer cells; therefore, the EZH2 has important significance in the field of targeted therapy of malignant diseases;
marek's Disease (MD) is a highly contagious neoplastic disease caused by Marek's Disease Virus (MDV) and is characterized by causing T cell tumors, and Meq (MDV Eco Q fragment-encoded protein) is an important oncogene in the MDV genome and is consistently expressed in latent tumor cells. Meq encodes a bZIP protein with a leucine zipper domain at the N-terminus and a proline-rich transactivation domain at the C-terminus;
however, the targeting relationship between the chicken EZH2 gene and the MDV genome is not described in the prior art.
Disclosure of Invention
Aiming at the existing problems, the invention aims to provide a method for researching the influence of chicken EZH2 gene on the neoplastic disease, and the method proves that the chicken EZH2 protein can inhibit the proliferation of MSB1 and influence the expression of MDV Meq and gB genes in the cell by researching the influence of chicken EZH2 gene on the neoplastic disease, thereby laying a foundation for further researching the action mechanism of the chicken EZH2 in MDV infection-induced tumor.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for researching influence of chicken EZH2 gene on neoplastic diseases comprises the steps of
S1, purifying chicken EZH2 prokaryotic protein to obtain a purified His tag EZH2 protein sample;
s2, co-culturing the purified chicken EZH2 prokaryotic expression protein and an MSB1 cell, and determining the influence of the EZH2 prokaryotic expression protein on the MSB1 cell proliferation;
s3, on the basis of the step S2, extracting total RNA of cells at different time points, detecting and analyzing the expression levels of the MDV related genes Meq and gB, and determining the influence of the EZH2 prokaryotic expression protein on the expression levels of the MDV related genes Meq and gB.
Preferably, the process for purifying chicken EZH2 prokaryotic protein described in step S1 comprises the following steps:
s101, carrying out induction culture on escherichia coli Rosetta (PET-32a-EZH2) containing a chicken EZH2 prokaryotic expression vector for 8 hours at 29 ℃ by using 1mmol of an inducer IPTG, collecting and weighing bacterial precipitates, and adding non-denaturing lysis solution according to the proportion of adding 4ml of non-denaturing lysis solution into each gram of bacterial precipitates;
s102, adding lysozyme until the final concentration is 1mg/ml, then fully and uniformly mixing, and carrying out ice-water bath for 30 min;
s103, ultrasonically cracking bacteria on ice, centrifuging for 30min at 4 ℃ under 10000g, and collecting the centrifuged supernatant in an EP tube and placing the tube on an ice water bath or ice;
s104, taking 1ml of uniformly mixed 50% BeyogoldTMHis-tag Purification Resin, centrifuging at 4 deg.C (1000g × 10s), discarding the storage solution, adding 0.5ml of non-denatured lysate into the gel in the Purification column, centrifuging at 4 deg.C, discarding the equilibrium solution, repeating the equilibrium for 2-3 times, and adding the supernatant of the bacterial lysate;
s105, collecting the flow-through liquid and repeatedly loading the flow-through liquid on the column for 3-5 times;
s106, then, taking down the cover at the bottom of the purification column to enable liquid in the column to naturally flow out due to the influence of gravity;
s107, adding 0.5ml of non-denaturing washing liquid each time, and then continuously washing the column for 5-6 times, wherein 20ul of washing liquid is collected each time;
s108, eluting the target protein for 6-10 times by using 0.5ml of non-denatured eluent each time to obtain a purified His label EZH2 protein sample.
Preferably, the specific application process of the chicken EZH2 prokaryotic expression protein co-cultured with the MSB1 cell in the step S2 comprises the following steps:
s201, diluting the centrifugally collected cells to 5.0 x 10 by using 1640 growth solution5Taking 100 mu L/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic proteins with different concentrations into each well of a treatment group to enable the final concentrations to be respectively 10, 20, 40 and 50 mu g/mL, adding pet-32a empty carrier protein with the equivalent volume and the corresponding concentration into a control group, setting 4 times for each concentration, setting a normal cell culture control without an antibody, and slightly shaking and uniformly mixing;
s202, after culturing for 24 hours and 48 hours in a 37 ℃ cell culture box, adding 10 mu l of CCK-8 reagent into each hole, after continuously culturing for 4 hours, adjusting the blank control to zero, and measuring OD on an enzyme labeling instrument450。
Preferably, the specific process of applying the chicken EZH2 prokaryotic expression protein described in the step S3 to MDV related genes comprises the following steps:
s301, diluting the centrifugally collected cells to 1.0X 10 by using 1640 growth solution6Taking 1 mL/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic protein with different concentrations into each well of a treatment group to enable the final concentration to be 50 mu g/mL, adding pet-32a empty carrier protein with the corresponding concentration in the same volume into a control group, and setting each concentration to be 4 times;
s302, continuously culturing for 24h and 48h, collecting cells, and extracting MSB1 cell total RNA;
s303, detecting the MDV related gene expression by using a real-time fluorescent quantitative PCR method.
Preferably, the MSB1 cell total RNA extraction process described in step S303 includes
(1) Collecting 2X 106-1×107MSB1 cell. Centrifuging at 2000 Xg for 5min, and discarding the supernatant;
(2) adding 400 μ l buffer solution R-I, repeatedly sucking with syringe equipped with 21-25 gauge needle for 8-10 times, and transferring into 1.5ml centrifuge tube;
(3) add 150. mu.l buffer R-II and vortex for 1min, centrifuge at 12000 Xg for 5min at 4 ℃;
(4) transferring the supernatant to a 1.5ml microcentrifuge tube, adding 250. mu.l of isopropanol and mixing thoroughly;
(5) placing the preparative column in a 2ml microcentrifuge tube, transferring the binding mixture from step (4) to the preparative tube, and centrifuging at 6000 Xg for 1 minute;
(6) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, adding 700. mu.l of BufferW1A into the preparation tube, and centrifuging for 1 minute at 12000 Xg;
(7) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, adding 700. mu.l Buffer W2, centrifuging at 12000 Xg for 1min, and washing once with 700. mu.l Buffer W2 by the same method;
(8) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, and centrifuging for 1 minute at 12000 Xg;
(9) placing the prepared tube into a clean 1.5ml centrifuge tube, adding 70-100 μ l Buffer TE or RNase-free water into the center of the prepared tube membrane, standing at room temperature for 1min, centrifuging at 12000 Xg for 1min, and eluting to obtain RNA.
Preferably, the real-time fluorescent quantitative PCR method for detecting MDV-related gene expression process of step S304 comprises
(1) Removing the genome DNA reaction;
(2) carrying out reverse transcription reaction of MSB1 cell total RNA;
(3) preparing Real Time reaction liquid by using an operation method of performing Real Time PCR reaction by using a TB Green Premix Ex Taq II method;
(4) performing Real Time PCR reaction;
(5) and calculating the expression level of the MDV related gene, and performing data analysis.
Preferably, in the Real Time PCR reaction process of step (3), a specific primer and a chicken internal reference gene are added into the reaction solution of the Real Time PCR reaction, and the specific primer and the chicken internal reference gene comprise
MDV Meq：F 5'-GTCCCCCCTCGATCTTTCTC-3′
R 5'-CGTCTGCTTCCTGCGTCTTC-3′；
gB：F 5'-ACCCCATTCGGTGGCTTTTC-3′
R 5'-GCGTCCAGTTGTCTGAGG-3′；
GAPDH：F 5'-TCTATCTCTTTGCTGGGACT-3′
R 5'-CTACAGCCAACTTTCATCAG-3′。
Preferably, the chicken EZH2 protein can inhibit the proliferation of MSB1 cell and affect the expression of MDV Meq and gB genes in the MSB1 cell.
Preferably, the chicken EZH2 protein has activity and can be applied to the research of action mechanism of the chicken in the chicken tumor diseases.
The invention has the beneficial effects that: the invention discloses a method for researching the influence of chicken EZH2 gene on tumor diseases, compared with the prior art, the improvement of the invention is as follows:
the invention uses MSB1 cell infected by MDV as model, purifies chicken EZH2 protein and co-cultures with MSB1 with different concentrations, and measures the influence of chicken EZH2 on MSB1 cell proliferation by CCK-8 method 24h and 48h after culture; simultaneously, after 50ug/mL of chicken EZH2 protein acts on MSB1 cells, total RNA of the cells is extracted at different time points, and the expression levels of MDV related genes Meq and gB are detected and analyzed by Real-time PCR;
the results show that: the chicken EZH2 with different concentrations of 10ug/mL, 20ug/mL, 40ug/mL and 50ug/mL acts on MSB1, and the CCK-8 method detects the MSB1 cell proliferation, and the result shows that the inhibition rate of the EZH2 protein group is not obviously different from that of an empty carrier protein control group (P is more than 0.05) at low concentration (10ug/mL and 20ug/mL), while the inhibition rate of the EZH2 protein group is obviously higher than that of the empty carrier protein control group (P is less than 0.05) at high concentration (40ug/mL and 50 ug/mL); after the 50ug/mLEZH2 protein and MSB1 cells are co-cultured for 24h, the MSB1 cell inhibition rate is not obviously different from that of a control group, and after 48h, the cell inhibition rate is obviously higher than that of the control group (P < 0.05); when the influence of chicken EZH2 on MDV related genes is researched, the relative expression quantity of the Meq and gB genes is obviously up-regulated (P is less than 0.05) in 24h and 48h compared with a control group, which indicates that chicken EZH2 prokaryotic expression protein plays a role in promoting the expression of the Meq and gB genes; the results prove that the chicken EZH2 protein can inhibit the proliferation of MSB1, and up-regulate the expression of MDV Meq and gB genes in the cell, thereby laying a foundation for further researching the action mechanism of the chicken EZH2 in MDV infection-induced tumors, and providing a theoretical basis for applying the EZH2 gene to the preparation of drugs for inhibiting the treatment of neoplastic diseases.
Drawings
FIG. 1 is a flow chart of the method for studying the effect of chicken EZH2 gene on neoplastic diseases.
FIG. 2 is a bar graph of cell proliferation potency assay of 48h of culture in example 1 of the present invention with different concentrations of EZH2 protein MSB 1.
FIG. 3 shows the OD of MSB1 cells after 124h and 48h in example of the present invention450Compare the figures.
FIG. 4 is a graph showing the detection of gB gene specificity in example 1 of the present invention.
FIG. 5 is a graph showing the results of specific detection of the Meq gene in example 1 of the present invention.
FIG. 6 is a histogram of the expression level detection of gB gene in example 1 of the present invention.
FIG. 7 is a histogram of the expression level detection of the Meq gene in example 1 of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the drawings and the embodiments.
Example 1:
referring to the attached figures 1-7, the method for studying the influence of chicken EZH2 gene on neoplastic diseases specifically comprises a material preparation process, a specific operation process and an experimental result analysis process, wherein:
the material preparation process comprises the following steps:
1. vector and protein purification
Prokaryotic expression vector PET-32a prokaryotic expression vector and host expression strain Rosetta thereof, prokaryotic expression vector PET-32a-EZH2 containing EZH2 target gene recombination is constructed and stored in animal epidemic disease and public safety laboratory (animal building 404) of Henan university of science and technology, and purification of chicken EZH2 prokaryotic expression protein is completed;
2. culture Medium for this experiment
The main reagents and media used in the study are listed in table 1 below:
table 1: culture medium for experiments
3. Main instrument
The main instruments of the experiment are as follows 2:
table 2: main instrument
4. Primer design and Synthesis
According to the mRNA sequences of EZH2(GenBank: Z48921.1), gB (GenBank: NC-002229.3), MDV Meq (GenBank: AB638843.1) and chicken internal reference Gene (GAPDH) (GenBank accession No.: NM-204305.1) which have been registered on GenBank; specific primers were designed according to primer 5.0 to avoid the formation of stable dimers or hairpin structures between primers that eventually lead to mismatches, and the sequences of primers synthesized by Service bio co ltd for amplification are shown in table 3:
table 3: amplification primers
(II) specific research process, comprising the steps of
S1, purifying chicken EZH2 prokaryotic protein
Beyogold is adopted in the experimentTMHis-tag Purification Resin for protein Purification:
s101, aiming at the target protein EZH2 protein, carrying out induction culture on escherichia coli Rosetta (PET-32a-EZH2) containing a chicken EZH2 prokaryotic expression vector for 8 hours by using 1mmol of an inducer IPTG (isopropyl-beta-thiogalactoside), and adding 4ml (2-5 ml) of non-denatured lysate according to the wet weight of bacterial precipitates per gram and the proportion of the non-denatured lysate, wherein the non-denatured lysate is added according to the operation aim of fully resuspending the bacteria;
s102, adding lysozyme to a final concentration of 1mg/ml, then fully and uniformly mixing, preparing a beaker, adding an ice-water mixture, and placing a sample in a water bath for 30 min;
wherein: lysozyme added: (ii) a non-denaturing lysate to volume ratio of 1: 10;
s103, ultrasonically cracking bacteria on ice by using the unpurified chicken EZH2 protein after water bath, which specifically comprises the following steps: centrifuging at 4 deg.C for 30min at 10000 Xg/min, collecting supernatant in EP tube, and placing in ice water bath or ice;
s104, sucking the uniformly mixed 50% Beyogold by using a liquid transfer gunTMPutting the solution of His-tag Purification Resin (1ml) in a 4 ℃ centrifuge for centrifugation at 1000 Xg/min for 10s, discarding the storage solution, then adding 0.5ml of non-denatured lysate into the gel in the Purification column, centrifuging at 4 ℃ centrifuge at 1000 Xg/min for 10s, discarding the storage solution, slowly repeating the balancing for 2-3 times, discarding the supernatant, and adding 4ml of bacterial lysate supernatant;
s105, collecting the flow-through liquid and repeatedly loading the flow-through liquid on the column for 3-5 times;
s106, then, taking down the cover at the bottom of the purification column to enable liquid in the column to naturally flow out due to the influence of gravity; 20ul of the downward flow liquid can be reserved for standby;
s107, adding 0.5ml of non-denaturing washing liquid each time, then continuously washing the column for 5-6 times, and collecting 20ul of washing liquid each time so as to be used for the next analysis and detection;
s108, eluting the target protein for 6-10 times, eluting with 0.5ml of non-denatured eluent each time, collecting the eluates each time into 10 marked EP tubes, and collecting the eluates containing purified His label EZH2 protein samples to obtain purified His label EZH2 protein samples;
note that: the purification process of the chicken EZH2 prokaryotic protein is carried out under the optimal expression condition of the EZH2 prokaryotic protein, wherein the optimal expression condition is as follows:
(1) the temperature was 29 °;
(2) the inducer concentration is 1.0mM IPTG;
(3) the induction time was 8 h.
S2, co-culturing the purified chicken EZH2 prokaryotic expression protein and MSB1 cells, and observing the influence of the EZH2 prokaryotic expression protein on the proliferation of the MSB1 cells:
s201, diluting the centrifugally collected cells to 5.0 x 10 by using 1640 growth solution5Taking 100 mu L/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic proteins with different concentrations into each well of a treatment group to enable the final concentrations to be respectively 10, 20, 40 and 50 mu g/mL, adding pet-32a empty carrier protein with the equivalent volume and the corresponding concentration into a control group, setting 4 times for each concentration, setting a normal cell culture control without an antibody, and slightly shaking and uniformly mixing;
s202, after culturing for 24 hours and 48 hours in a 37 ℃ cell culture box, adding 10 mu l of CCK-8 reagent into each hole, after continuously culturing for 4 hours, adjusting the blank control to zero, and measuring OD on an enzyme labeling instrument450。
S3, on the basis of the step S2, extracting total RNA of cells at different time points, detecting and analyzing the expression levels of MDV related genes Meq and gB, and determining the influence of the EZH2 prokaryotic expression protein on the expression levels of the MDV related genes Meq and gB, wherein the method specifically comprises the following steps:
s301, diluting the centrifugally collected cells to 1.0X 10 by using 1640 growth solution6Taking 1 mL/mL, inoculating the cells in a 96-well cell culture plate, adding chicken EZH2 prokaryotic protein with different concentrations into each well of a treatment group to enable the final concentration to be 50 mu g/mL, adding pet-32a empty carrier protein with the corresponding concentration in the same volume into a control group, and setting each concentration to be 4 times;
s302, inoculating the cells into 6 holes, wherein each hole is 2mL and contains about 106 cells, adding EZH2 and empty carrier protein respectively to make the total concentration of the cells be 50ug/mL, and incubating the cells for 48h at 37 ℃;
s303, collecting and culturing 24h and 48h cells in a 6-well plate respectively, finally extracting MSB1 cell total RNA, wherein the detailed steps of extracting MSB1 cell total RNA under different culture conditions are as follows:
(1) collecting 2X 106-1×107MSB1 cells of (1), centrifuged at 2000 Xg for 5minClock, abandon the supernatant fluid;
(2) add 400. mu.l buffer R-I, aspirate repeatedly 8-10 times with a syringe equipped with a 21-25 gauge needle, and transfer to a 1.5ml centrifuge tube (provided in the kit).
(3) Add 150. mu.l buffer R-II and vortex for 1min, centrifuge at 12000 Xg for 5min at 4 ℃;
(4) transferring the supernatant to a 1.5ml microcentrifuge tube, adding 250. mu.l of isopropanol and mixing thoroughly;
(5) placing the preparative column in a 2ml microcentrifuge tube, transferring the binding mixture from step (4) to the preparative tube, and centrifuging at 6000 Xg for 1 minute;
(7) discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, adding 700. mu.l of BufferW1A into the preparation tube, and centrifuging for 1 minute at 12000 Xg;
(8) the filtrate was discarded, the preparation tube was put back into a 2ml microcentrifuge tube, and 700. mu.l of Buffer W2 was added thereto, and the mixture was centrifuged at 12000 Xg for 1 minute, followed by washing once with 700. mu.l of Buffer W2 in the same manner.
(9) Discarding the filtrate, placing the preparation tube back into a 2ml microcentrifuge tube, and centrifuging for 1 minute at 12000 Xg;
(10) the preparation tube was placed into a clean 1.5ml centrifuge tube (provided in the kit) and 70-100. mu.l Buffer TE or RNase-free water was added to the center of the preparation tube membrane. Standing at room temperature for 1min, centrifuging at 12000 Xg for 1min, and eluting to obtain RNA;
s304, detecting MDV related gene expression by using a real-time fluorescent quantitative PCR method, which specifically comprises the following steps:
(1) reactions for removing genomic DNA
Respectively extracting total RNA of MSB1 cells co-cultured by different proteins, and removing the genome in the extracted total RNA; preparing a reaction mixed solution on ice according to the following components in the following table 4, wherein in order to ensure the accuracy of the preparation of the reaction solution in the process, during each reaction, Master Mix is prepared according to more than 2 reaction quantities, then the Master Mix is subpackaged into each reaction tube, and finally an RNA sample is added, wherein the reaction solutions are as shown in the table 4:
TABLE 4 genomic DNA removal reaction solution
Reaction conditions are as follows: storing at 42 deg.C for 2min and 4 deg.C;
(2) carrying out reverse transcription reaction of MSB1 cell total RNA;
in order to ensure the accuracy of the preparation of the reaction solution, during each reaction, Master Mix is prepared according to more than 2 reaction quantities, then 10 μ l of Master Mix is dispensed into each reaction tube, after gentle and uniform mixing, reverse transcription reaction is immediately carried out, the reaction solution is prepared on an ice water mixture, and the reaction solution is shown in the following table 5:
table 5: reaction solution for reverse transcription
Reaction conditions are as follows: storing at 37 deg.C for 15min, 85 deg.C for 5sec, and 4 deg.C;
(3)Real time PCR
the procedure for Real Time PCR reaction was performed according to TB Green Premix Ex Taq II (Tli RNaseH Plus) (Code No. RR820A/B); a PCR reaction solution (prepared in an ice-water mixture) was prepared with the following components as shown in Table 6:
table 6: reaction solution of Real Time reaction
(4) Performing Real Time PCR reaction;
two-step PCR amplification standard procedure:
stage 1: pre-denaturation: 95 deg.C, 30 seconds, one cycle
Stage 2: and (3) PCR reaction: 95 ℃ for 5 seconds; 60 ℃ for 60 seconds; 40 cycles
(5) Calculating the expression level of MDV related gene, and performing data analysis
By using 2-△△CtThe method calculates the relative expression of MDV Meq and gB genes in each group of MSB1 cells.
(III) analysis procedure of experimental results
1. Effect of chicken EZH2 prokaryotic expression protein on proliferation of MSB1 cell
1.1, detecting the proliferation rate of MSB1 cells by a CCK-8 method after chicken EZH2 prokaryotic expression proteins with different concentrations enter MSB1 cells for 48 hours, as shown in figure 2;
from fig. 2, it can be observed that after different concentrations of EZH2 prokaryotic expression protein are added into MSB1 cells, at low concentrations (10ug/mL and 20ug/mL), the inhibition rate of EZH2 protein group is not significantly different from that of the empty carrier protein control group (P >0.05), while at high concentrations (40ug/mL and 50ug/mL), the inhibition rate of EZH2 protein group is significantly higher than that of the empty carrier protein control group (P <0.05), thereby demonstrating that EZH2 prokaryotic expression protein can inhibit the proliferation of MSB1 cells and has concentration dependence;
1.2 Effect of prokaryotic expression protein of chicken EZH2 on proliferation of MSB1 cells at different times
The chicken EZH2 prokaryotic expression protein enters the MSB1 cell for 24h at different time, and the proliferation of the MSB1 cell is detected by a CCK-8 method after 48h, as shown in figure 3;
as can be seen from fig. 3, after adding the prokaryotic expression protein of chicken EZH2 into MSB1 cells at different concentrations, the inhibition rate of EZH2 proteome was not significantly different from that of the empty carrier protein control group at 24h (P >0.05), while at 48h, EZH2 proteome significantly inhibited MSB1 cell proliferation (P <0.01) compared with the empty carrier protein;
2. detection of MDV Meq, gB expression level by Real-time PCR
2.1 characterization of the dissolution Curve
(1) After chicken EZH2 prokaryotic expression protein is added into MSB1 cells for 24h, detecting the gene expression level of MDV related gene gB by using Real-timePCR, and performing dissolution curve identification, as shown in figure 4;
(2) after chicken EZH2 prokaryotic expression protein is added into MSB1 cells for 24h, the gene expression level of MDV related gene Meq is detected by using Real-timePCR, and a dissolution curve is identified and shown in figure 5;
analysis of the solubility curves of gB and Meq amplification products (see figure 4 and figure 5) shows that the solubility curves of all target genes are only single peaks and have no impurity peaks, and that only one amplification exists in the Real-time PCR process, so that the established Real-time PCR method is good in specificity.
2.2 changes in the relative expression level of the MDV gB Gene
(1) After chicken EZH2 prokaryotic expression protein is added into MSB1 cells for 24h and 48h, Real-timePCR is used for detecting the gene expression quantity of the MDV related gene gB, and the expression quantity is shown in figure 6; the results showed that the relative expression amount of MDV-related gene gB of MSB1 cells to which EZH2 recombinant protein was added was significantly up-regulated (P <0.01) at 24h and 48h compared to the control group, indicating that the expression of the gene of gB was promoted in MSB1 cells co-cultured with chicken EZH2 compared to the control group;
2.3 changes in the relative expression level of the MDV Meq Gene
After chicken EZH2 prokaryotic expression protein is added into MSB1 cells for 24h and 48h, Real-timePCR is utilized to detect the gene expression quantity of MDV related gene Meq, and is shown in figure 7; the results showed that the relative expression amount of MDV-related gene Meq of MSB1 cells to which EZH2 protein was added was significantly up-regulated (P <0.01) at 24h and 48h compared to the control group, indicating that the expression of the gene of Meq was promoted in MSB1 cells co-cultured with chicken EZH2 compared to the control group.
By our findings in this study:
(1) the chicken EZH2 prokaryotic expression protein influences the proliferation capability of MSB1 cells, can inhibit the proliferation of MSB1 cells, and has certain concentration dependence;
(2) the influence of the chicken EZH2 protein on the MDV related gene expression in MSB1 cells is detected by using a fluorescent quantitative PCR technology, and the chicken EZH2 protein shows up-regulation on the MDV related gB gene and Meq gene expression level relative to an empty carrier proteome.
(3) The prokaryotic expression protein of chicken EZH2 can be applied to the research of the action mechanism of the prokaryotic expression protein in chicken tumor diseases.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.