CN111549069A - Lentiviral vector carrying human ROCK2 gene and construction and application thereof - Google Patents

Lentiviral vector carrying human ROCK2 gene and construction and application thereof Download PDF

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CN111549069A
CN111549069A CN202010443534.7A CN202010443534A CN111549069A CN 111549069 A CN111549069 A CN 111549069A CN 202010443534 A CN202010443534 A CN 202010443534A CN 111549069 A CN111549069 A CN 111549069A
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rock2 gene
rock2
human rock2
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姚文娟
祁妍
梁秀瑛
赵荷艳
刘兆国
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Nantong University
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Abstract

The invention discloses a lentiviral vector carrying a human ROCK2 gene, and construction and application thereof, and belongs to the technical field of biomedicine. The lentiviral vector carrying the human ROCK2 gene is obtained by inserting the human ROCK2 gene into the AgeI enzyme cutting site of the vector GV287-Ubi-MCS-3FLAG-SV 40-EGFP; the nucleotide sequence of the human ROCK2 gene is shown as SEQ ID NO. 1. According to the invention, the transfection efficiency of eukaryotic cells can be greatly improved by constructing the lentivirus carrying the ROCK2 gene, and the ROCK2 gene can integrate an endothelial cell genome, stably express for a long time, and screen to obtain a cell strain stably expressing the ROCK2 gene.

Description

Lentiviral vector carrying human ROCK2 gene and construction and application thereof
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a lentiviral vector carrying a human ROCK2 gene as well as construction and application thereof.
Background
Rho-associated coiled coil protein kinase (ROCK) is the effector of the small GTP-binding protein Rho. ROCK was first discovered in 1996 as the first Rho downstream effector, and the Rho-associated coiled coil kinase (ROCK) family includes ROCK1 and ROCK2, which share about 65% of the total amino acid sequence identity and their kinase domains are about 92% similar. It has been found that the mRNA and protein of ROCK1 are highly expressed in lung, liver, spleen, kidney and testis, while ROCK2 is abundantly expressed in brain and heart. ROCK2 is predominantly localized to the cytoplasm, and in contrast to the intracellular distribution of ROCK1, which is associated with vimentin and actin stress fibers, studies have not been well established. ROCK plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and migration, cell division and gene expression.
Studies have shown that ROCK activation is involved in many cardiovascular diseases, such as hypertension and atherosclerosis. Clinically used unique ROCK inhibitor fasudil shows satisfactory effects on aspects of controlling blood pressure, regulating arteriosclerotic coronary artery diseases, treating heart failure and the like, but fasudil is a non-selective inhibitor, has an inhibiting effect on two subtypes of ROCK, and is very important for screening out ROCK inhibitors with strong specificity or high selectivity. However, the lack of an effective in vitro screening model makes it difficult to obtain selective ROCK inhibitors.
The traditional construction of gene over-expression eukaryotic cell strain adopts recombinant plasmid transfection and G418 screening, and the following problems are easy to occur: the liposome transfection efficiency is low, and the electrotransformation cost is expensive; the screening period is at least three months, and excessive manpower and financial resources are consumed; the screened false positive rate is high, and the situation that the false positive can grow under the G418 screening pressure is easy to occur; the selected cell line often fails to stably express the gene for a long period of time.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide a lentiviral vector carrying the human ROCK2 gene, which can be used for screening a vascular endothelial cell model capable of stably expressing ROCK2, and which can be used for screening compounds capable of selectively inhibiting ROCK 2.
In order to achieve the purpose, the invention adopts the following technical scheme:
a lentiviral vector carrying a human ROCK2 gene is obtained by inserting the human ROCK2 gene into the AgeI enzyme cutting site of a vector GV287-Ubi-MCS-3FLAG-SV 40-EGFP;
the nucleotide sequence of the human ROCK2 gene is as follows (SEQ ID NO. 1):
ATGAGCCGGCCCCCGCCGACGGGGAAAATGCCCGGCGCCCCCGAGACCGCGCCGGGGGACGGGGCAGGCGCGAGCCGCCAGAGGAAGCTGGAGGCGCTGATCCGAGACCCTCGCTCCCCCATCAACGTGGAGAGCTTGCTGGATGGCTTAAATTCCTTGGTCCTTGATTTAGATTTTCCTGCTTTGAGGAAAAACAAGAACATAGATAATTTCTTAAATAGATATGAGAAAATTGTGAAAAAAATCAGAGGTCTACAGATGAAGGCAGAAGACTATGATGTTGTAAAAGTTATTGGAAGAGGTGCTTTTGGTGAAGTGCAGTTGGTTCGTCACAAGGCATCGCAGAAGGTTTATGCTATGAAGCTTCTTAGTAAGTTTGAAATGATAAAAAGATCAGATTCTGCCTTTTTTTGGGAAGAAAGAGATATTATGGCCTTTGCCAATAGCCCCTGGGTGGTTCAGCTTTTTTATGCCTTTCAAGATGATAGGTATCTGTACATGGTAATGGAGTACATGCCTGGTGGAGACCTTGTAAACCTTATGAGTAATTATGATGTGCCTGAAAAATGGGCCAAATTTTACACTGCTGAAGTTGTTCTTGCTCTGGATGCAATACACTCCATGGGTTTAATACACAGAGATGTGAAGCCTGACAACATGCTCTTGGATAAACATGGACATCTAAAATTAGCAGATTTTGGCACGTGTATGAAGATGGATGAAACAGGCATGGTACATTGTGATACAGCAGTTGGAACACCGGATTATATATCACCTGAGGTTCTGAAATCACAAGGGGGTGATGGTTTCTATGGGCGAGAATGTGATTGGTGGTCTGTAGGTGTTTTCCTTTATGAGATGCTAGTGGGGGATACTCCATTTTATGCGGATTCACTTGTAGGAACATATAGCAAAATTATGGATCATAAGAATTCACTGTGTTTCCCTGAAGATGCAGAAATTTCCAAACATGCAAAGAATCTCATCTGTGCTTTCTTAACAGATAGGGAGGTACGACTTGGGAGAAATGGGGTGGAAGAAATCAGACAGCATCCTTTCTTTAAGAATGATCAGTGGCATTGGGATAACATAAGAGAAACGGCAGCTCCTGTAGTACCTGAACTCAGCAGTGACATAGACAGCAGCAATTTCGATGACATTGAAGATGACAAAGGAGATGTAGAAACCTTCCCAATTCCTAAAGCTTTTGTTGGAAATCAGCTGCCTTTCATCGGATTTACCTACTATAGAGAAAATTTATTATTAAGTGACTCTCCATCTTGTAGAGAAACTGATTCCATACAATCAAGGAAAAATGAAGAAAGTCAAGAGATTCAGAAAAAACTGTATACATTAGAAGAACATCTTAGCAATGAGATGCAAGCCAAAGAGGAACTGGAACAGAAGTGCAAATCTGTTAATACTCGCCTAGAAAAAACAGCAAAGGAGCTAGAAGAGGAGATTACCTTACGGAAAAGTGTGGAATCAGCATTAAGACAGTTAGAAAGAGAAAAGGCGCTTCTTCAGCACAAAAATGCAGAATATCAGAGGAAAGCTGATCATGAAGCAGACAAAAAACGAAATTTGGAAAATGATGTTAACAGCTTAAAAGATCAACTTGAAGATTTGAAAAAAAGAAATCAAAACTCTCAAATATCCACTGAGAAAGTGAATCAACTCCAGAGACAACTGGATGAAACCAATGCTTTACTGCGAACAGAGTCTGATACTGCAGCCCGGTTAAGGAAAACCCAGGCAGAAAGTTCAAAACAGATTCAGCAGCTGGAATCTAACAATAGAGATCTACAAGATAAAAACTGCCTGCTGGAGACTGCCAAGTTAAAACTTGAAAAGGAATTTATCAATCTTCAGTCAGCTCTAGAATCTGAAAGGAGGGATCGAACCCATGGATCAGAGATAATTAATGATTTACAAGGTAGAATATGTGGCCTAGAAGAAGATTTAAAGAACGGCAAAATCTTACTAGCGAAAGTAGAACTGGAGAAGAGACAACTTCAGGAGAGATTTACTGATTTGGAAAAGGAAAAAAGCAACATGGAAATAGATATGACATACCAACTAAAAGTTATACAGCAGAGCCTAGAACAAGAAGAAGCTGAACATAAGGCCACAAAGGCACGACTAGCAGATAAAAATAAGATCTATGAGTCCATCGAAGAAGCCAAATCAGAAGCCATGAAAGAAATGGAGAAGAAGCTCTTGGAGGAAAGAACTTTAAAACAGAAAGTGGAGAACCTATTGCTAGAAGCTGAGAAAAGATGTTCTCTATTAGACTGTGACCTCAAACAGTCACAGCAGAAAATAAATGAGCTCCTTAAACAGAAAGATGTGCTAAATGAGGATGTTAGAAACCTGACATTAAAAATAGAGCAAGAAACTCAGAAGCGCTGCCTTACACAAAATGACCTGAAGATGCAAACACAACAGGTTAACACACTAAAAATGTCAGAAAAGCAGTTAAAGCAAGAAAATAACCATCTCATGGAAATGAAAATGAACTTGGAAAAACAAAATGCTGAACTTCGAAAAGAACGTCAGGATGCAGATGGGCAAATGAAAGAGCTCCAGGATCAGCTCGAAGCAGAACAGTATTTCTCAACCCTTTATAAAACACAAGTTAGGGAGCTTAAAGAAGAATGTGAAGAAAAGACCAAACTTGGTAAAGAATTGCAGCAGAAGAAACAGGAATTACAGGATGAACGGGACTCTTTGGCTGCCCAACTGGAGATCACCTTGACCAAAGCAGATTCTGAGCAACTGGCTCGTTCAATTGCTGAAGAACAATATTCTGATTTGGAAAAAGAGAAGATCATGAAAGAGCTGGAGATCAAAGAGATGATGGCTAGACACAAACAGGAACTTACGGAAAAAGATGCTACAATTGCTTCTCTTGAGGAAACTAATAGGACACTAACTAGTGATGTTGCCAATCTTGCAAATGAGAAAGAAGAATTAAATAACAAATTGAAAGATGTTCAAGAGCAACTGTCAAGATTGAAAGATGAAGAAATAAGCGCAGCAGCTATTAAAGCACAGTTTGAGAAGCAGCTATTAACAGAAAGAACACTCAAAACTCAAGCTGTGAATAAGTTGGCTGAGATCATGAATCGAAAAGAACCTGTCAAGCGTGGTAATGACACAGATGTGCGGAGAAAAGAGAAGGAGAATAGAAAGCTACATATGGAGCTTAAATCTGAACGTGAGAAATTGACCCAGCAGATGATCAAGTATCAGAAAGAACTGAATGAAATGCAGGCACAAATAGCTGAAGAGAGCCAGATTCGAATTGAACTGCAGATGACATTGGACAGTAAAGACAGTGACATTGAGCAGCTGCGGTCACAACTCCAAGCCTTGCATATTGGTCTGGATAGTTCCAGTATAGGCAGTGGACCAGGGGATGCTGAGGCAGATGATGGGTTTCCAGAATCAAGATTAGAAGGATGGCTTTCATTGCCTGTACGAAACAACACTAAGAAATTTGGATGGGTTAAAAAGTATGTGATTGTAAGCAGTAAGAAGATTCTTTTCTATGACAGTGAACAAGATAAAGAACAATCCAATCCTTACATGGTTTTAGATATAGACAAGTTATTTCATGTCCGACCAGTTACACAGACAGATGTGTATAGAGCAGATGCTAAAGAAATTCCAAGGATATTCCAGATTCTGTATGCCAATGAAGGAGAAAGTAAGAAGGAACAAGAATTTCCAGTGGAGCCAGTTGGAGAAAAATCTAATTATATTTGCCACAAGGGACATGAGTTTATTCCTACTCTTTATCATTTCCCAACCAACTGTGAGGCTTGTATGAAGCCCCTGTGGCACATGTTTAAGCCTCCTCCTGCTTTGGAGTGCCGCCGTTGCCATATTAAGTGTCATAAAGATCATATGGACAAAAAGGAGGAGATTATAGCACCTTGCAAAGTATATTATGATATTTCAACGGCAAAGAATCTGTTATTACTAGCAAATTCTACAGAAGAGCAGCAGAAGTGGGTTAGTCGGTTGGTGAAAAAGATACCTAAAAAGCCCCCAGCTCCAGACCCTTTTGCCCGATCATCTCCTAGAACTTCAATGAAGATACAGCAAAACCAGTCTATTAGACGGCCAAGTCGACAGCTTGCCCCAAACAAACCTAGCTAA。
the construction method of the lentivirus vector comprises the following steps:
step 1, fishing a human ROCK2 gene from cDNA of a cell containing the human ROCK2 gene by using a high fidelity enzyme KOD-PCR method, and then amplifying the human ROCK2 gene by using primers to obtain an amplified fragment of the human ROCK2 gene, wherein the two ends of the amplified fragment contain enzyme cutting sites;
and 2, connecting the gene segment obtained in the step 2 to a GV287-Ubi-MCS-3FLAG-SV40-EGFP vector, transforming a bacterial competent cell DH5a by using a connecting product, screening positive clones, extracting plasmids, and completing construction of a lentiviral vector.
A lentivirus carrying the human ROCK2 gene is obtained by transfecting 293T cells with the above lentivirus vector.
An endothelial cell line stably expressing human ROCK2 gene was obtained by infecting HUVEC cells with the above lentivirus carrying human ROCK2 gene and passaging and screening the cells in a medium containing puromycin at a final concentration of 1. mu.g/mL.
The application of the endothelial cell strain in screening ROCK2 inhibitors.
According to the invention, the transfection efficiency of eukaryotic cells can be greatly improved by constructing the lentivirus carrying the ROCK2 gene, and the ROCK2 gene can integrate an endothelial cell genome, stably express for a long time, and screen to obtain a cell strain stably expressing the ROCK2 gene.
Drawings
FIG. 1 shows the result of PCR amplification of the target gene in example 1. The left lane is Marker and the right lane is the target gene. The Marker sequentially comprises the following components from top to bottom: 8kb, 6kb, 4kb, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250 bp.
FIG. 2 shows the result of electrophoresis of the cleavage product of example 1. Lane 1 is Marker, Lane 2 is the carrier enzyme-cleaved product, Lane 3 is the carrier not enzyme-cleaved, Marker is from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250 bp.
FIG. 3 shows the results of PCR identification of positive clones in example 1. Lane 1 is a negative control (ddH)2O), lane 2 is a negative control (no-load self-ligation control), lane 3 is a positive control (GAPDH), lane 4 is Marker, and lanes 5-12 are transformants ROCK 21-8. The Marker sequentially comprises 5kb, 4kb, 3kb, 2000bp, 1500bp, 1000bp, 750bp, 500bp and 250bp from top to bottom.
FIG. 4 is a Real-time PCR curve for the lentivirus titer determination in example 2.
FIG. 5 shows the results of Western blot analysis of endothelial cells in example 3.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
The following examples employ the following main materials:
lentiviral vectors: GV287-Ubi-MCS-3FLAG-SV40-EGFP was purchased from Invitrogen.
293T cells, packaging cells for lentivirus, anchorage-dependent epithelioid cells, growth medium DMEM (10% FBS). The adherent cells grow and proliferate to form a monolayer of cells after culture.
Coli strain DH5 α, for amplification of lentiviral vectors and helper packaging vector plasmids.
Primary cells: human Umbilical Vein Endothelial Cells (HUVECs).
Example 1
The construction of a lentiviral vector carrying a human ROCK2 gene comprises the following specific steps:
1. a target gene is extracted from cDNA of a cell containing the target gene by a high fidelity enzyme KOD-PCR method, and then a PCR product is purified and recovered.
The primer sequences are as follows:
ROCK2-FGAGGATCCCCGGGTACCGGTCGCCACCATGAGCCGGCCCCCGCCGA(SEQ ID NO.2)
ROCK2-RTCCTTGTAGTCCATACCTCAGAAATCAAAAACATCGTCATCAT(SEQ ID NO.3)
the sequence to which the vector GV287 was ligated is underlined, the black sequence is the primer for PCR ROCK2 gene, the PCR product is 4167bp, and the results of electrophoresis are shown in FIG. 1.
The PCR reaction system for PCR amplification of target gene fragments is as follows:
reagent Volume of μ L
ddH2O 16.2μL
10x KOD-buffer 2.5μL
dNTPs 2.5μL
MgSO4 1.5μL
Primer(F)(10μM) 0.3μL
Primer(R)(10μM) 0.3μL
KOD-Hi-Fi enzymes 0.3μL
cDNA template 2μL
Total volume 25μL
2. Carrying out enzyme digestion on the target vector, wherein the cloning site is AgeI, the enzyme digestion reaction condition is 37 ℃ and 2h, carrying out 16 ℃ connection overnight after enzyme digestion product electrophoresis recovery, and transforming the connection product into the bacterial competent cell DH5 a. And (3) carrying out colony PCR identification on the grown clones, and then sequencing and comparing and analyzing the clones with positive PCR identification, wherein the correctly compared clones are the successfully constructed target plasmids. The correct clones were aligned, plasmids were extracted and stored at-20 ℃ for future use.
The electrophoresis result of the cleavage product is shown in FIG. 2.
PCR identification positive clone and sequencing identification
Identifying the primer: ROCK2-F ATGAGCCGGCCCCCGCCGACGGGGAAAA (SEQ ID NO.4)
ROCK2-R AAGTGAATCCGCATAAAATGGAG(SEQ ID NO.5)
The PCR product size of the positive transformant is 1000bp, and the electrophoresis chart is shown in 3.
Because the CDS sequence of ROCK2 is too long to 4167bp, only about 1000bp can be sequenced in one sequencing reaction, 5 segments of sequencing primers are divided for sequencing, and the sequencing primers are as follows:
ROCK2-1F CTGGCCGTTTTTGGCTTTTTT(SEQ ID NO.6)
ROCK2-2F GTAGGTGTTTTCCTTTATGAGAT(SEQ ID NO.7)
ROCK2-3F CTCCAGAGACAACTGGATGAAACCAAT(SEQ ID NO.8)
ROCK2-4F AACTTGGAAAAACAAAATGCTG(SEQ ID NO.9)
ROCK2-5R TCCTTGTAGTCCATACCTCAGAAA(SEQ ID NO.10)。
the sequencing result shows that the plasmid is successfully constructed.
Example 2
The slow virus package carrying the human ROCK2 gene comprises the following specific steps:
1. 24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and adjusted to a cell density of 1.2 × 10 in a medium containing 10% serum7The cells/20 mL were re-seeded in 15cm cell culture dishes at 37 ℃ with 5% CO2Culturing in an incubator. Can be used for transfection after the cell density reaches 70-80% in 24 h. The cell state is critical for virus packaging and therefore it is desirable to ensure good cell state and a low number of passages.
2. Cell culture medium was changed to serum-free medium 2h before transfection.
3. Each prepared DNA solution (20. mu.g of GV287 vector, 1520. mu.g of pHelper 1.0 vector, 10. mu.g of pHelper 2.0 vector) was added to a sterilized centrifuge tube, and the resulting mixture was mixed with an irrelevant volume of Opti-MEM, adjusted to a total volume of 2.5mL, and incubated at room temperature for 5 minutes.
4. Lipofectamine 2000 was gently shaken, 100. mu.L of Lipofectamine 2000 was mixed with 2.4mL of Opti-MEM in another tube, and incubated at room temperature for 5 minutes.
5. The diluted DNA and the diluted Lipofectamine 2000 were mixed, and the mixture was gently inverted and mixed without shaking. Mixing must be within 5 minutes.
6. After mixing, incubation was performed at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution.
7. Transferring the mixture of DNA and Lipofectamine 2000 to 293T cell culture medium, mixing, and culturing at 37 deg.C with 5% CO2Culturing in a cell culture box.
8. After 8h of incubation, the medium containing the transfection mixture was decanted, 20mL of PBS was added to each flask, the flask was gently shaken side-to-side to wash the residual transfection mixture, and then decanted.
9. 25mL of cell culture medium containing 10% serum was added to each flask of cells at 37 ℃ with 5% CO2The incubator was allowed to incubate for 48 hours.
10. Supernatants from 293T cells were collected 48 hours after transfection (which could be 0 hours after transfection).
11. Cell debris was removed by centrifugation at 12000rpm for 10min at 4 ℃.
12. The supernatant was filtered through a 0.45 μm filter into a 40mL ultracentrifuge tube.
13. The crude virus extract sample was added to the filter cup and the lid was closed. The filter cup is inserted into the permeate collection tube.
14. After the combination, the balance is well made and placed on the rotating head.
15. Centrifuge at 12000rpm to the desired virus concentration volume. The time required is generally from 10 to 15 minutes.
16. After centrifugation is completed, the centrifuge is removed and the filter cup is separated from the lower filtrate collection cup.
17. The filter cup was inverted over the sample collection cup.
18. The centrifugal force does not exceed 1000rpm and the time is 2 minutes. Too high a rotational speed can result in loss of sample. The filter cup is removed from the sample collection cup. The sample in the sample collecting cup is virus concentrated solution.
19. Removing the virus concentrated solution, subpackaging and storing in a virus tube, and storing at-80 ℃ for a long time. One of the branches was used for virus biological titer determination.
Lentivirus titer assay carrying the human ROCK2 gene:
first, sample preparation
1. The day before the assay, 293T cells were passaged and 1 × 10 was added to each 24-well5Individual cells, volume 500 μ L;
2. the next day, 7-10 sterile Ep tubes were prepared, and 90. mu.L of medium (DMEM + 10% FBS) was added to each tube;
3. add 10. mu.L of the virus stock solution to be measured into the first tube, mix well, add 10. mu.L into the second tube. Continuing the same operation until the last tube;
4. the desired cell wells were selected and 90. mu.L of medium was aspirated. The diluted virus solution was added. Adding 5% CO at 37 deg.C2Culturing in an incubator;
after 5.24 hours, 500. mu.L of fresh medium was added. Carefully operating, not blowing up the cells, and maintaining the normal growth of the cells;
after 6.4 days, RNA was extracted in preparation for RT-qPCR.
Description of the drawings:
adding 10 mu L of virus stock solution into the first Ep tube, and recording as 1E +1 mu L;
the first tenfold dilution was performed in the second Ep tube, and the resulting virus stock solution was 1/10 in the first Ep tube, and was recorded as 1E + 0. mu.L;
the third Ep tube is subjected to the second tenfold dilution, and the obtained virus stock solution is 1/10 in the second Ep tube and is recorded as 1E-1 mu L;
and so on …
A sixteenth tenfold dilution was performed in the seventh Ep tube and the resulting virus stock was 1/10 in the sixth Ep tube, noted as 1E-5. mu.L.
Second, total RNA extraction
Description of the drawings: the procedures were performed according to the TRIZOL instruction manual of InvitroGen, and all were RNase-free procedures.
1. The cell supernatant was removed, 1mL of TRIZOL was added to each well, and the mixture was left to stand at room temperature for 5min and then transferred to another new 1.5mL eppendorf tube.
2. Add 200. mu.L chloroform to each tube, shake vigorously for 15s, and stand at room temperature for 15 min.
Centrifuge at 12000rpm for 15min at 3.4 ℃.
4. The supernatant from each tube was aspirated into another new 1.5mL eppendorf tube. Adding equal volume of pre-cooled isopropanol at-20 deg.C, mixing, and precipitating at-20 deg.C for 10 min.
After centrifugation at 12000rpm for 10min at 5.4 ℃ the supernatant was removed.
6. At least 1mL of 75% ethanol pre-cooled at 4 ℃ was added, and the pellet and centrifuge tube walls were washed.
Centrifuge at 12000rpm for 5min at 7.4 deg.C, and discard the supernatant.
Centrifuge again at 12000rpm for 5min at 8.4 deg.C, remove residual liquid by suction, and dry at room temperature (without complete drying).
9. mu.L of RNase-free water was added to dissolve completely, and the concentration of extracted RNA was determined by UV analysis.
Thirdly, reverse transcription of RNA to obtain cDNA
M-MLV reverse transcriptase and dNTPs were purchased from PROMEGA. Oligo dT was purchased from Shanghai Producer. RNase-free was purchased from AxyGen.
Description of the drawings: the procedures were carried out according to the M-MLV protocol of PromeGa, and all procedures were RNase-free.
The specific operation is as follows:
1. mu.L OliGo dT (0.5. mu.G/. mu.L) and 2.0. mu.G Total RNA were added to the PCR vials, supplemented with DEPC-H2O to 9. mu.L. Mixing, centrifuging, and bathing at 70 deg.C for 10 min. Immediately thereafter, the plate was inserted into an ice-water bath at 0 ℃ to anneal OliGo dT and the template.
2. The required amount of reagent was calculated from the number of reaction tubes in the proportions shown in the following table. The M-MLV enzyme and the like were mixed well on ice to obtain an RT reaction solution.
Figure BDA0002504801960000091
3. mu.L of RT reaction solution was added to each reaction tube, mixed well and centrifuged.
RT reaction was completed after 1h at 42 ℃ followed by 10min treatment at 70 ℃ to inactivate RT enzyme.
5. The resulting RT reaction product, cDNA, can be used immediately for PCR or stored at-80 ℃ for later use.
Four, Real-time PCR detection
Real-time PCR was performed on 7900 of ABI. SYBR Master mix is from ABI.
1. The reaction system was prepared in the following proportions:
amount of reagent added per well
SYBR master mix:2.5μL
Upstream primer (10 μ M): 0.15 μ L
Downstream primer (10 μ M): 0.15 μ L
cDNA(100ng/μL)：2.2μL
Figure BDA0002504801960000092
2. The procedure was set to two step Real-Time quantitation. The pre-expansion is carried out for 40 cycles at 95 ℃ for 15S, and then the annealing elongation is carried out for 40S at 95 ℃ for each step and 5S for 30S. Each time reading the absorbance value during the extension phase.
3. A melting curve was prepared. After the PCR was completed, the mixture was incubated at 95 ℃ for 1 min. Then cooled to 55 ℃ to allow the DNA double strands to be fully bound. Starting at 55 ℃ to 95 ℃, each step was increased by 0.5 ℃ and held for 30S while absorbance was read.
The Real-time PCR curve is shown in FIG. 4.
And (3) analyzing the Ct value and the expression quantity of a sample group after virus infection with different concentrations:
Figure BDA0002504801960000101
in this titer test, 10-4The Ct value of the samples in the μ L group and Con group was different and was considered to be 10-4Viral particles were present in the μ L group samples. Assuming that the group of samples contains at least 1 virus particle, the titer of the virus is:
1/(1.00E-04)*20＝2.00E+5TU/μL＝2.00E+8TU/mL。
example 3
Endothelial cell line screening for stably expressing human ROCK2 Gene
Culturing HUVEC cells with good growth state to logarithmic growth phase, digesting with 0.25% pancreatin for 37-2 min, washing with PBS for 1 time, and counting cell plating in 6-well plate (50 ten thousand/well/2 mL). And after 24 hours of plating, the plating rate of the cells reaches about 60%, 1mL of the virus wrapped in the early stage is added into each well, the virus titer is 2 x 10^8, the virus is changed into a fresh culture solution after being infected by the virus for 48 hours, 1 mug/mL of puromycin is added to stably screen for 1 month, and then the well-grown cells are collected for Western blot identification and detection.
The Western blot comprises the following detailed experimental steps:
1. extraction of total cellular protein: cells in logarithmic growth phase are digested for 2min by 0.25% pancreatin, washed for 2 times by PBS, transferred to a clean 1.5mLEP tube, marked, precooled low-temperature centrifuge is added to 4 ℃, 400 mu L of protein lysate is added to each tube, continuously stirred and cracked for 10min by a gun head on ice, centrifuged for 20min at 4 ℃ and 12000rpm, and supernatant is carefully sucked into another clean 1.5mLEP tube by a micropipettor.
2. Protein denaturation: adding 1/4 volumes of 5 loading buffers into 1 volume of protein to prepare a mixed solution, boiling the mixed solution in 100 ℃ boiling water for 10min, recalculating the protein concentration, and storing the mixed solution at-80 ℃.
3. A10% SDS-PAGE gel was prepared and loaded at 60. mu.g/lane.
4. Electrophoresis: running glue at 80v for 30min, then running glue at 120v for 90min (when the glue is separated slightly by observing the change of each strip, the blue dye reaches near the glue bottom, stopping electrophoresis, black-to-black electrodes, red-to-red electrodes)
5. Wet-to-membrane (this operation was performed in membrane transfer solution — wet transfer): the film was transferred on ice at a constant pressure of 100V for 2h, (transfer box white vs. red, black vs. black) until the corresponding Marker was transferred to the PVDF film.
6. Sealing of
Preparing TBST containing 5% of skimmed milk:
TBST: 1mL Tween20, 100mL 10X TBS and 900mL double distilled water, and mixing uniformly, preferably preparing the mixture just before use.
TBST of 5% skim milk: 2.5g of skimmed milk powder was added to 50mL of TBST, dissolved and stored at 4 ℃. When in use, the temperature is recovered to room temperature, and the dosage is covered by the membrane surface, thus the disposable mask is used for one time.
Sealing for 1h at room temperature on a shaking bed, and washing the membrane for 15min and X3 times by TBST.
7. Antibody incubation
Antibody dilution: primary antibody was diluted proportionally with blocking solution of TBST milk.
Primary antibody incubation: spreading a sealing film at the bottom of the box, dropping antibody, spreading PVDF film with the front surface (protein surface) upward and the other surface (antibody), spreading a sealing film, standing overnight at 4 deg.C (shaking table), and incubating at room temperature for 60 min.
Washing: the PVDF membrane was removed and washed 15min x 3 times with TBST (shaking table).
And (3) incubating the fluorescent secondary antibody (after the fluorescent secondary antibody is added, the incubation of the secondary antibody and the washing and developing stages at the later stage need to be carried out in a dark place): the secondary antibody is added according to the proportion of 1: diluted 5000, prepared in 5mL of 5% TBST milk (according to the secondary antibody instructions) and incubated for 2h at room temperature (shaker).
Washing a secondary antibody: TBST wash 3 times, 10min x 3 times.
8. Fluorescent light emission color development
And placing the films in a fluorescence imager in sequence for exposure, and storing pictures.
The machine used was the Odyssey two-color infrared fluorescence imaging system from LI-COR, usa.
The results are shown in FIG. 5. The cell model can stably express ROCK2 gene, Western blot result proves that the expression quantity of ROCK2 of the cell strain is 29 times of that of a normal group, and the cell strain can be applied to screening selective ROCK2 inhibitor.
Sequence listing
<110> university of southeast Tong
<120> lentiviral vector carrying human ROCK2 gene, and construction and application thereof
<130>20200522
<160>12
<170>SIPOSequenceListing 1.0
<210>1
<211>4167
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atgagccggc ccccgccgac ggggaaaatg cccggcgccc ccgagaccgc gccgggggac 60
ggggcaggcg cgagccgcca gaggaagctg gaggcgctga tccgagaccc tcgctccccc 120
atcaacgtgg agagcttgct ggatggctta aattccttgg tccttgattt agattttcct 180
gctttgagga aaaacaagaa catagataat ttcttaaata gatatgagaa aattgtgaaa 240
aaaatcagag gtctacagat gaaggcagaa gactatgatg ttgtaaaagt tattggaaga 300
ggtgcttttg gtgaagtgca gttggttcgt cacaaggcat cgcagaaggt ttatgctatg 360
aagcttctta gtaagtttga aatgataaaa agatcagatt ctgccttttt ttgggaagaa 420
agagatatta tggcctttgc caatagcccc tgggtggttc agctttttta tgcctttcaa 480
gatgataggt atctgtacat ggtaatggag tacatgcctg gtggagacct tgtaaacctt 540
atgagtaatt atgatgtgcc tgaaaaatgg gccaaatttt acactgctga agttgttctt 600
gctctggatg caatacactc catgggttta atacacagag atgtgaagcc tgacaacatg 660
ctcttggata aacatggaca tctaaaatta gcagattttg gcacgtgtat gaagatggat 720
gaaacaggca tggtacattg tgatacagca gttggaacac cggattatat atcacctgag 780
gttctgaaat cacaaggggg tgatggtttc tatgggcgag aatgtgattg gtggtctgta 840
ggtgttttcc tttatgagat gctagtgggg gatactccat tttatgcgga ttcacttgta 900
ggaacatata gcaaaattat ggatcataag aattcactgt gtttccctga agatgcagaa 960
atttccaaac atgcaaagaa tctcatctgt gctttcttaa cagataggga ggtacgactt 1020
gggagaaatg gggtggaaga aatcagacag catcctttct ttaagaatga tcagtggcat 1080
tgggataaca taagagaaac ggcagctcct gtagtacctg aactcagcag tgacatagac 1140
agcagcaatt tcgatgacat tgaagatgac aaaggagatg tagaaacctt cccaattcct 1200
aaagcttttg ttggaaatca gctgcctttc atcggattta cctactatag agaaaattta 1260
ttattaagtg actctccatc ttgtagagaa actgattcca tacaatcaag gaaaaatgaa 1320
gaaagtcaag agattcagaa aaaactgtat acattagaag aacatcttag caatgagatg 1380
caagccaaag aggaactgga acagaagtgc aaatctgtta atactcgcct agaaaaaaca 1440
gcaaaggagc tagaagagga gattacctta cggaaaagtg tggaatcagc attaagacag 1500
ttagaaagag aaaaggcgct tcttcagcac aaaaatgcag aatatcagag gaaagctgat 1560
catgaagcag acaaaaaacg aaatttggaa aatgatgtta acagcttaaa agatcaactt 1620
gaagatttga aaaaaagaaa tcaaaactct caaatatcca ctgagaaagt gaatcaactc 1680
cagagacaac tggatgaaac caatgcttta ctgcgaacag agtctgatac tgcagcccgg 1740
ttaaggaaaa cccaggcaga aagttcaaaa cagattcagc agctggaatc taacaataga 1800
gatctacaag ataaaaactg cctgctggag actgccaagt taaaacttga aaaggaattt 1860
atcaatcttc agtcagctct agaatctgaa aggagggatc gaacccatgg atcagagata 1920
attaatgatt tacaaggtag aatatgtggc ctagaagaag atttaaagaa cggcaaaatc 1980
ttactagcga aagtagaact ggagaagaga caacttcagg agagatttac tgatttggaa 2040
aaggaaaaaa gcaacatgga aatagatatg acataccaac taaaagttat acagcagagc 2100
ctagaacaag aagaagctga acataaggcc acaaaggcac gactagcaga taaaaataag 2160
atctatgagt ccatcgaaga agccaaatca gaagccatga aagaaatgga gaagaagctc 2220
ttggaggaaa gaactttaaa acagaaagtg gagaacctat tgctagaagc tgagaaaaga 2280
tgttctctat tagactgtga cctcaaacag tcacagcaga aaataaatga gctccttaaa 2340
cagaaagatg tgctaaatga ggatgttaga aacctgacat taaaaataga gcaagaaact 2400
cagaagcgct gccttacaca aaatgacctg aagatgcaaa cacaacaggt taacacacta 2460
aaaatgtcag aaaagcagtt aaagcaagaa aataaccatc tcatggaaat gaaaatgaac 2520
ttggaaaaac aaaatgctga acttcgaaaa gaacgtcagg atgcagatgg gcaaatgaaa 2580
gagctccagg atcagctcga agcagaacag tatttctcaa ccctttataa aacacaagtt 2640
agggagctta aagaagaatg tgaagaaaag accaaacttg gtaaagaatt gcagcagaag 2700
aaacaggaat tacaggatga acgggactct ttggctgccc aactggagat caccttgacc 2760
aaagcagatt ctgagcaact ggctcgttca attgctgaag aacaatattc tgatttggaa 2820
aaagagaaga tcatgaaaga gctggagatc aaagagatga tggctagaca caaacaggaa 2880
cttacggaaa aagatgctac aattgcttct cttgaggaaa ctaataggac actaactagt 2940
gatgttgcca atcttgcaaa tgagaaagaa gaattaaata acaaattgaa agatgttcaa 3000
gagcaactgt caagattgaa agatgaagaa ataagcgcag cagctattaa agcacagttt 3060
gagaagcagc tattaacaga aagaacactc aaaactcaag ctgtgaataa gttggctgag 3120
atcatgaatc gaaaagaacc tgtcaagcgt ggtaatgaca cagatgtgcg gagaaaagag 3180
aaggagaata gaaagctaca tatggagctt aaatctgaac gtgagaaatt gacccagcag 3240
atgatcaagt atcagaaaga actgaatgaa atgcaggcac aaatagctga agagagccag 3300
attcgaattg aactgcagat gacattggac agtaaagaca gtgacattga gcagctgcgg 3360
tcacaactcc aagccttgca tattggtctg gatagttcca gtataggcag tggaccaggg 3420
gatgctgagg cagatgatgg gtttccagaa tcaagattag aaggatggct ttcattgcct 3480
gtacgaaaca acactaagaa atttggatgg gttaaaaagt atgtgattgt aagcagtaag 3540
aagattcttt tctatgacag tgaacaagat aaagaacaat ccaatcctta catggtttta 3600
gatatagaca agttatttca tgtccgacca gttacacaga cagatgtgta tagagcagat 3660
gctaaagaaa ttccaaggat attccagatt ctgtatgcca atgaaggaga aagtaagaag 3720
gaacaagaat ttccagtgga gccagttgga gaaaaatcta attatatttg ccacaaggga 3780
catgagttta ttcctactct ttatcatttc ccaaccaact gtgaggcttg tatgaagccc 3840
ctgtggcaca tgtttaagcc tcctcctgct ttggagtgcc gccgttgcca tattaagtgt 3900
cataaagatc atatggacaa aaaggaggag attatagcac cttgcaaagt atattatgat 3960
atttcaacgg caaagaatct gttattacta gcaaattcta cagaagagca gcagaagtgg 4020
gttagtcggt tggtgaaaaa gatacctaaa aagcccccag ctccagaccc ttttgcccga 4080
tcatctccta gaacttcaat gaagatacag caaaaccagt ctattagacg gccaagtcga 4140
cagcttgccc caaacaaacc tagctaa4167
<210>2
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gaggatcccc gggtaccggt cgccaccatg agccggcccc cgccga 46
<210>3
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tccttgtagt ccatacctca gaaatcaaaa acatcgtcat cat 43
<210>4
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atgagccggc ccccgccgac ggggaaaa 28
<210>5
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
aagtgaatcc gcataaaatg gag 23
<210>6
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ctggccgttt ttggcttttt t 21
<210>7
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gtaggtgttt tcctttatga gat 23
<210>8
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ctccagagac aactggatga aaccaat 27
<210>9
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
aacttggaaa aacaaaatgc tg 22
<210>10
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
tccttgtagt ccatacctca gaaa 24
<210>12
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
aattccgtgg tgttgtcg 18
<210>13
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
aaggtccgct ggattgag 18

Claims (5)

1. A lentiviral vector carrying a human ROCK2 gene, wherein: human ROCK2 gene is inserted into AgeI enzyme cutting site of vector GV287-Ubi-MCS-3FLAG-SV 40-EGFP;
the nucleotide sequence of the human ROCK2 gene is shown as SEQ ID NO. 1.
2. The method of constructing a lentiviral vector of claim 1, wherein: the method comprises the following steps:
step 1, fishing a human ROCK2 gene from cDNA of a cell containing the human ROCK2 gene by using a high fidelity enzyme KOD-PCR method, and then amplifying the human ROCK2 gene by using primers to obtain an amplified fragment of the human ROCK2 gene, wherein the two ends of the amplified fragment contain enzyme cutting sites;
and 2, connecting the gene segment obtained in the step 2 to a GV287-Ubi-MCS-3FLAG-SV40-EGFP vector, transforming a bacterial competent cell DH5a by using a connecting product, screening positive clones, extracting plasmids, and completing construction of a lentiviral vector.
3. A lentivirus carrying the human ROCK2 gene, which is characterized in that: obtained by transfecting 293T cells with the lentiviral vector of claim 1.
4. An endothelial cell line stably expressing the human ROCK2 gene, comprising: HUVEC cells infected with the lentivirus of claim 3 were passaged through selection in medium containing puromycin at a final concentration of 1 μ g/mL.
5. Use of the endothelial cell strain of claim 4 for screening for a ROCK2 inhibitor.
CN202010443534.7A 2020-05-22 2020-05-22 Lentiviral vector carrying human ROCK2 gene and construction and application thereof Pending CN111549069A (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN102212535A (en) * 2011-04-08 2011-10-12 单云峰 Construction and identification of over-expression lentivirus vector of rat GSK-3 beta (Glycogen Synthase Kinase-3 beta) target gene
CN109266683A (en) * 2018-10-12 2019-01-25 中南大学湘雅二医院 A kind of slow virus recombinant vector and its preparation method and application comprising E4BP4 gene

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Publication number Priority date Publication date Assignee Title
CN102212535A (en) * 2011-04-08 2011-10-12 单云峰 Construction and identification of over-expression lentivirus vector of rat GSK-3 beta (Glycogen Synthase Kinase-3 beta) target gene
CN109266683A (en) * 2018-10-12 2019-01-25 中南大学湘雅二医院 A kind of slow virus recombinant vector and its preparation method and application comprising E4BP4 gene

Non-Patent Citations (4)

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Title
CHEN,J.: "Homo sapiens mRNA for ROCK2 (ROCK2 gene)", 《GENBANK：LT978485.1》 *
HE-MING ZHOU ET AL.: "Overlapping and unique roles played by ROCK1 and 2 in the modulation of coding and long noncoding RNA expression", 《BMC GENOMICS 》 *
吴邦财等: "携带siRNA的慢病毒载体抑制自发性高血压大鼠阴茎海绵体平滑肌细胞S1PR3的表达", 《中华男科学杂志》 *
李晓江等: "ROCK-Ⅱ基因shRNA质粒表达载体的构建与鉴定", 《中国老年学杂志》 *

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Application publication date: 20200818