CN112210556A - Group of shRNA for targeted interference of IL-33 expression, recombinant adenovirus vector, and construction method and application thereof - Google Patents
Group of shRNA for targeted interference of IL-33 expression, recombinant adenovirus vector, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a group of shRNA and recombinant adenovirus vectors for targeted interference of IL-33 gene or protein expression, and a construction method and application thereof, belonging to the technical field of genetic engineering. The invention provides a group of shRNAs for targeted interference of IL-33 gene or protein expression, wherein the corresponding nucleotide sequences are shown as SEQ ID NOs: 1 to 3. The invention also provides a group of recombinant adenovirus vectors respectively containing 3 shRNA sequences. The three recombinant adenovirus vectors and shuttle plasmids infect eukaryotic cells under the conditions of liposome and DMEM culture medium respectively, and 3 recombinant adenoviruses capable of interfering IL-33 gene expression are packaged. The three recombinant adenoviruses are used together, so that the expression of IL-33 genes and proteins in a mouse body can be effectively reduced, and a technical support is provided for the deep research of a mechanism of IL-33 as a hepatic fibrosis treatment target.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a group of shRNA for targeted interference of IL-33 expression, a recombinant adenovirus vector, and a construction method and application thereof.
Background
RNA interference technology is a new developed genetic engineering technology in the last ten years, can inhibit the expression of target genes with high specificity, and provides a valuable tool for researching the biological function of a certain gene and personalized treatment. The adenovirus vector infected cell can be transferred into target gene, and is widely used for gene silencing research due to its high efficiency and stable transfection characteristics. There are many members of the adenovirus family, but only two of them are used to construct adenovirus vectors, all belonging to subfamily C, one being adenovirus type 2 (Ad2) and the other being adenovirus type 5 (Ad 5). Both sides of the adenovirus contain ITR, and the replication initiation site is contained, so that the adenovirus can replicate normally in cells. There are currently four adenoviral vector packaging systems: homologous recombination, Ad-Easy method, AdMax packaging system and Ad5/F35MaxTm packaging system.
Interleukin 33 (Interleukin-33, IL-33) is a cytokine of a super family member of interleukin 1, and is originally found to express IL-33 in a large amount in liver, stomach, kidney and lung, but is mainly produced by endothelial cells, epithelial cells, fibroblast-like cells and myofibroblasts, and can promote the immunoregulation effect of Th2 type cells. IL-33 binds to ST2 receptor and transmits activated signal to cells, and then downstream signal is activated through a myeloid differentiation factor 88(MyD88) signal channel, so that NF-kB complex in the cells is released, and the cells are damaged.
Since the discovery of IL-33, its role in the treatment of chronic liver injury has been considerably studied and its pathogenesis is mainly linked to the ability of IL-33 to activate hepatic stellate cells and thus promote liver fibrosis. The diversity and complexity of their biological roles has revealed that they may be involved in the process of disease development and progression through different mechanisms. IL-33 has been considered as an alert signal in the early stage of liver injury and thus can be used as a novel liver injury marker to prevent liver diseases; in addition, IL-33 can also be used for treating some chronic liver injury diseases, but excessive use can also cause liver fibrosis and produce side effects. While removal of IL-33 may also reduce liver fibrosis damage, IL-33 appears to be a new target for treatment of liver fibrosis, and the implementation of these strategies is yet to be further investigated in view of the source of liver damage.
The potential feasibility of IL-33 as a hepatic fibrosis treatment drug can be deeply explored by interfering the expression of IL-33 gene, but a convenient and effective mouse IL-33 expression interference technology is not available at present.
Disclosure of Invention
In view of the above, the present invention aims to provide a group of shRNAs which target and interfere with the expression of IL-33 gene or protein, and can effectively reduce the expression level of IL-33 gene.
The invention aims to provide a group of recombinant adenovirus vectors for targeted interference of IL-33 gene or protein expression and a construction method thereof, wherein the recombinant adenovirus vectors comprise shRNA for targeted interference of IL-33 gene or protein expression and can down-regulate the expression level of IL-33 in mice.
The invention also aims to provide application of the group of shRNA or recombinant adenovirus vectors in preparation of medicines for treating hepatic fibrosis injury.
The invention provides a group of shRNAs for targeting and interfering IL-33 gene or protein expression, which comprises shIL-33(250), shIL-33(456) and shIL-33 (594);
the nucleotide sequence of the shIL-33(250) is shown as SEQ ID NO:1 is shown in the specification;
the nucleotide sequence of the shIL-33(456) is shown in SEQ ID NO:2 is shown in the specification;
the nucleotide sequence of the shIL-33(594) is shown in SEQ ID NO:3, respectively.
The invention provides a group of recombinant adenovirus vectors for targeted interference of IL-33 gene or protein expression, which comprise shRNA.
Preferably, the shRNA is inserted into multiple cloning sites of pDC316-ZsGreen1-shRNA adenovirus vectors by shIL-33(250), shIL-33(456) and shIL-33(594) respectively.
Preferably, the multiple cloning site is the cleavage site of PstI and BamHI.
The invention provides a construction method of the recombinant adenovirus vector, which comprises the following steps:
1) adding PstI and BamHI cleavage sites to both ends of shIL-33(250), shIL-33(456) or shIL-33(594) in the shRNA, and removing the first G base of the BamHI cleavage site to obtain shIL-33(250), shIL-33(456) or shIL-33(594) containing cleavage sites at both ends;
2) carrying out double enzyme digestion on the adenovirus vector pDC316-ZsGreen1-shRNA by using restriction endonucleases PstI and BamHI to obtain a linearized pDC316-ZsGreen1 vector;
3) carrying out T4 connection on the linearized pDC316-ZsGreen1 vector obtained in the step 2) and the shIL-33(250), shIL-33(456) or shIL-33(594) with the enzyme cutting sites at two ends obtained in the step 1) to obtain three recombinant adenovirus vectors which are respectively named as: pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456), pDC316-ZsGreen1-shIL-33 (594).
Preferably, after the T4 is connected, the method further comprises the step of verifying three recombinant adenovirus expression plasmids by BamHI single enzyme digestion;
there is no chronological restriction between step 1) and step 2).
The invention provides a recombinant adenovirus packaging method, which is prepared by the following steps:
A. mixing pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) and shuttle plasmid pBHGlox (delta) E1 and 3Cre obtained by the construction method according to the mass ratio of 5-6: 3-4, and mixing the mixture with a DMEM culture medium to prepare a mixed solution Mix 1;
B. preparing a mixed solution Mix2 by using liposome Lipofectamine TM2000 and DMEM medium;
C. uniformly mixing the Mix1 and Mix2, and standing to obtain an invasion liquid;
D. and C, infecting eukaryotic cells with the infection solution obtained in the step C, observing fluorescence after 48h, and collecting viruses after 10 days to obtain recombinant adenovirus shIL-33(250), recombinant adenovirus shIL-33(456) and recombinant adenovirus shIL-33 (594).
Preferably, in the Mix1, the total mass concentration of pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) and shuttle plasmid pBHGlox (delta) E1,3Cre is 2 μ g/mL.
The invention provides recombinant adenoviruses prepared by the recombinant adenovirus packaging method, wherein each recombinant adenovirus comprises shRNA for the targeted interference of IL-33 gene or protein expression.
The invention provides an application of the shRNA, the recombinant adenovirus vector or the recombinant adenovirus in preparing a medicament for treating hepatic fibrosis injury.
The invention provides a group of shRNAs for targeting and interfering IL-33 gene or protein expression, which comprises shIL-33(250), shIL-33(456) and shIL-33 (594); the nucleotide sequence of the shIL-33(250) is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the shIL-33(456) is shown in SEQ ID NO:2 is shown in the specification; the nucleotide sequence of the shIL-33(594) is shown in SEQ ID NO:3, respectively. WesternBlot and immunohistochemical detection experiment results show that the three shRNAs provided by the invention can effectively reduce the expression level of the IL-33 gene in a mouse by being matched together.
The invention provides a group of recombinant adenovirus vectors for targeted interference of IL-33 gene or protein expression, which comprise shRNA. The recombinant adenovirus vector is based on three pDC316-ZsGreen1-ShRNA adenovirus interference plasmids, 3 shRNA sequences of mouse IL-33 genes are respectively introduced into a multiple cloning site, and the plasmid also carries green fluorescent protein, so that positive cells can be observed conveniently.
The invention also provides a recombinant adenovirus interfering the expression of the IL-33 gene of the mouse, the recombinant adenovirus is injected into the mouse through tail vein to realize the effective down-regulation of the IL-33 expression quantity of the mouse, and the invention provides technical support for the deep research of the mechanism of the IL-33 as the fibrosis treatment target.
Drawings
FIG. 1 shows the double restriction enzyme linearization of pDC316-ZsGreen1-shRNA vector; 1: vector pDC316-ZsGreen 1-shRNA; m1: marker1, DL 2000 Marker; m2: marker2, 1kb DNA Ladder;
FIG. 2 shows the restriction enzyme identification of the recombinant plasmid pDC316-ZsGreen 1-shIL-33; m: marker, 1kb DNA Ladder; 1: shIL-33(250) original plasmid; 2: shIL-33(250) BamHI single enzyme digestion verification, wherein the enzyme digestion is not cut because the starting position of the 5' end has less G basic groups; 3: shIL-33(250) PstI single enzyme digestion verification, and cutting out a single strip; 4: shIL-33(250) is verified by BamHI and PstI double enzyme digestion, and a single strip is cut out; 5: shIL-33(456) original plasmid; 6: shIL-33(456) BamHI single enzyme digestion verification; 7: shIL-33(456) PstI single enzyme digestion verification; 8: shIL-33(456) was verified by double digestion with BamHI and PstI; 9: shIL-33(594) original plasmid; 10: shIL-33(594) BamHI single enzyme digestion verification; 11: shIL-33(594) PstI single enzyme digestion verification; 12: shIL-33(594) was verified by double digestion with BamHI and PstI;
FIG. 3 shows the expression of green fluorescent protein after plasmid cotransfection of HEK293 cells; fig. 3A to 3C: transfecting 48h images by an optical microscope and a fluorescence microscope, wherein the cells express a small amount of GFP, and the expression amount of GFP is increased (200X) along with the increase of the infection time; fig. 3D to 3F: transfection of 10d fluorescence microscopy images, almost complete detachment of cells, lesion formation, detoxification, expression of large amounts of GFP protein (100 ×);
FIG. 4 is Western Blot identification of interference effect of recombinant adenovirus;
FIG. 5 shows immunohistochemical identification of interference effects of recombinant adenovirus; FIG. 5A is a graph showing the results that mature hepatocytes in the control group expressed IL-33 (400X); FIG. 5B shows that the expression level of IL-33 in cells in the adenovirus group was decreased, and that the interference efficiency was satisfactory (400X);
FIG. 6 shows the results of different shRNAs interfering with the expression level of IL-33.
Detailed Description
The invention provides a group of shRNAs for targeting and interfering IL-33 gene or protein expression, which comprises shIL-33(250), shIL-33(456) and shIL-33 (594). The mouse IL-33 sequence (GI: 1041274026) was looked up in the NCBI database and three shRNA sequences were designed at positions 250, 456, 594. The nucleotide sequence of the shIL-33(250) is shown as SEQ ID NO:1 (ggcctatccacgggattctaggcgaacctagaatcccgtggataggctttttgatcaaaaagcctatccacgggattctaggttcgcctagaatcccgtggataggccctgca); the nucleotide sequence of the shIL-33(456) is shown in SEQ ID NO:2 (ggcaagaccaggtgctactacgcgaacgtagtagcacctggtcttgctttttgatcaaaaagcaagaccaggtgctactacgttcgcgtagtagcacctggtcttgccctgca); the nucleotide sequence of the shIL-33(594) is shown in SEQ ID NO:3 (gggactactccgtggagcttcacgaatgaagctccacggagtagtcctttttgatcaaaaaggactactccgtggagcttcattcgtgaagctccacggagtagtcccctgca). The source of the shRNA in the present invention is not particularly limited, and a source of shRNA known in the art may be used. In the examples of the present invention, the shRNA was synthesized by bioengineering company.
The shRNA can also be obtained by connecting the obtained single-stranded DNA into double strands by adopting gradient annealing PCR. The reaction procedure for the gradient PCR is as follows:
reacting at 95 ℃ for 5min, at 85 ℃ for 5min, at 75 ℃ for 5min, at 65 ℃ for 5min, at 55 ℃ for 5min, at 40 ℃ for 5min, at 30 ℃ for 5min, and terminating the reaction at 25 ℃.
The reaction system of the gradient PCR is as follows:
| name of reagent | Volume (μ L) |
| 10× |
1 |
| Upstream sequence (100. mu. mol) | 1 |
| Downstream sequence (100. mu. mol) | 1 |
| ddH2O | 7 |
The invention provides a group of recombinant adenovirus vectors for targeted interference of IL-33 gene or protein expression, which comprise shRNA. The recombinant adenovirus vector is preferably shIL-33(250), shIL-33(456) and shIL-33(594) in the shRNA, which are respectively inserted into a polyclonal site of the pDC316-ZsGreen1-shRNA adenovirus vector. The multiple cloning site is preferably the cleavage site for PstI and BamHI. The pDC316-ZsGreen1-shRNA adenovirus vector was obtained from Microbix Biosystems, Inc.
The invention provides a construction method of the recombinant adenovirus vector, which comprises the following steps:
1) adding PstI and BamHI cleavage sites to both ends of shIL-33(250), shIL-33(456) or shIL-33(594) in the shRNA, and removing the first G base of the BamHI cleavage site to obtain shIL-33(250), shIL-33(456) or shIL-33(594) containing cleavage sites at both ends;
2) carrying out double enzyme digestion on the adenovirus vector pDC316-ZsGreen1-shRNA by using restriction endonucleases PstI and BamHI to obtain a linearized pDC316-ZsGreen1 vector;
3) carrying out T4 connection on the linearized pDC316-ZsGreen1 vector obtained in the step 2) and the shIL-33(250), shIL-33(456) or shIL-33(594) which is obtained in the step 1) and contains enzyme cutting sites at two ends to obtain three recombinant adenovirus expression plasmids which are respectively named as: pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456), pDC316-ZsGreen1-shIL-33 (594);
there is no chronological restriction between step 1) and step 2).
According to the invention, PstI and BamHI cleavage sites are added at two ends of shIL-33(250), shIL-33(456) or shIL-33(594) in shRNA, and then the first G base of the BamHI cleavage site is removed, so that shIL-33(250), shIL-33(456) or shIL-33(594) containing cleavage sites at two ends is obtained.
In the present invention, it is preferable to add PstI cleavage site (ctgcag) to the 5 'end of shIL-33(250), shIL-33(456) or shIL-33(594), and it is preferable to add BamHI cleavage site to the 3' end of shIL-33(250), shIL-33(456) or shIL-33(594) to remove the first G base of BamHI cleavage site (ggatcc) so that BamHI cannot enzymatically cleave the new vector after the fragment is ligated to the vector. The method for adding the PstI enzyme cutting site and the PstI enzyme cutting site is preferably obtained by a gene synthesis method.
The invention uses restriction endonucleases PstI and BamHI to carry out double enzyme digestion on adenovirus vector pDC316-ZsGreen1-shRNA, and obtains a linearized pDC316-ZsGreen1 vector.
In the present invention, the reaction conditions of the enzyme digestion are preferably as follows: incubation was carried out at 37 ℃ for 4 h. The reaction system of the enzyme digestion is as follows:
| reagent | Volume (μ L) |
| CutSmart Buffer(10×) | 5 |
| |
|
| Endonuclease BamHI | |
| 2 | |
| pDC316 plasmid | 10μg |
| ddH2O | Make up to 50. mu.L |
After obtaining a linearized pDC316-ZsGreen1 vector, the invention connects the linearized pDC316-ZsGreen1 vector with shIL-33(250), shIL-33(456) or shIL-33(594) containing enzyme cutting sites at two ends thereof by T4 to obtain three recombinant adenovirus expression plasmids.
In the present invention, the linking system is preferably as follows:
| reagent | Volume (μ L) |
| DNA Ligase Buffer(10×) | 1 |
| pDC316 plasmid after digestion (100ng) | 1 |
| shIL-33 |
1 |
| |
1 |
| ddH2O | Complement 10 |
The reaction conditions for the ligation are preferably as follows: ligation was carried out overnight at 16 ℃.
In the present invention, after said T4 ligation, preferably, BamHI is further included for single enzyme digestion verification of three recombinant adenovirus plasmids. The BamHI single enzyme digested vector is verified by agarose gel electrophoresis, and positive clone is obtained without incision.
The invention provides a recombinant adenovirus packaging method, which comprises the following steps:
A. pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) obtained by the construction method and a shuttle plasmid pBHGlox (delta) E1,3Cre are mixed according to the proportion of 5-6: 3-4, and mixing with a DMEM culture medium to prepare a mixed solution Mix 1;
B. preparing a mixed solution Mix2 by using liposome Lipofectamine TM2000 and DMEM medium;
C. uniformly mixing the Mix1 and Mix2, and standing to obtain an invasion liquid;
D. infecting the eukaryotic cells with the infection liquid obtained in the step 3), observing fluorescence after 48h, and collecting viruses after 10 days to obtain recombinant adenovirus shIL-33(250), recombinant adenovirus shIL-33(456) and recombinant adenovirus shIL-33 (594).
The invention relates to pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) and shuttle plasmid pBHGlox (delta) E1,3Cre obtained by the construction method according to the ratio of 5-6: 3-4, and mixing with a DMEM medium to prepare a mixed solution Mix 1.
In the present invention, the mass ratio of pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) to shuttle plasmid pBHGlox (delta) E1,3Cre is preferably 5.5: 3.5. In the present invention, in the Mix1, the total mass concentration of pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) and shuttle plasmid pBHGlox (delta) E1,3Cre is preferably 2 μ g/mL.
The invention prepares the liposome Lipofectamine TM2000 and DMEM medium into mixed liquid Mix 2.
In the invention, the volume ratio of the liposome Lipofectamine TM2000 to the DMEM medium in the Mix2 is 3: 125.
After obtaining Mix1 and Mix2, Mix1 and Mix2 are mixed evenly and are kept still to obtain the staining solution.
In the invention, the volume ratio of Mix1 to Mix2 is 1: 1. The standing time is preferably 10-12 min.
After the infection liquid is obtained, the infection liquid is infected into eukaryotic cells, fluorescence is observed after 48 hours, viruses are collected after 10 days, and the recombinant adenovirus shIL-33(250), the recombinant adenovirus shIL-33(456) and the recombinant adenovirus shIL-33(594) are obtained.
In the present invention, the eukaryotic cell is preferably a HEK293 cell. The HEK293 cells are preferably seeded in 6-well plates to give 5 cells per well×105And (4) respectively. The dosage of the staining solution is preferably 2ml/5 x 105A eukaryotic cell. After infection, cultivation is preferably carried out. The conditions for the culture are preferably as follows: 5% CO at 37 ℃2Culturing in an incubator, and changing a DMEM medium containing 10% fetal calf serum after 12 h.
In the invention, the method for collecting the virus preferably comprises the steps of repeatedly freezing and thawing the cultured cells and the culture medium for 3 times, collecting the crushed cells and the culture medium, centrifuging at 3000rpm for 10min, and collecting the supernatant to obtain the shRNA recombinant adenovirus interfering the IL-33 gene of the mouse.
The invention provides recombinant adenoviruses prepared by the recombinant adenovirus packaging method, wherein each recombinant adenovirus comprises shRNA (short hairpin ribonucleic acid) which is targeted to interfere IL-33 gene or protein expression and is respectively marked as recombinant adenovirus shIL-33(250), recombinant adenovirus shIL-33(456) and recombinant adenovirus shIL-33 (594).
The invention provides an application of the shRNA, the recombinant adenovirus vector or the recombinant adenovirus in preparing a medicament for treating hepatic fibrosis injury.
In the invention, the shRNA, the recombinant adenovirus vector or the recombinant adenovirus are all three shRNA, recombinant adenovirus vector or recombinant adenovirus mixed according to equal copy number to prepare the medicine. The prepared medicament is injected into a mouse through tail vein, can effectively reduce the expression of IL-33 of the mouse, and provides technical support for deeply researching the mechanism of IL-33 as a fibrosis treatment target.
The following examples are provided to describe in detail a group of shRNA targeted to interfere with IL-33 expression, recombinant adenovirus vectors, and their construction methods and applications, but they should not be construed as limiting the scope of the present invention.
In the following examples, the experimental procedures not specifying the specific conditions were generally carried out according to the experimental procedures commonly used by those skilled in the art to which the present invention pertains, as described in "molecular biology laboratory Manual of Fine text" (edited by F.M. Osber, R.E. Kingston, J.G. Sedman et al, Massachusetts, Shujon, Beijing: scientific Press, 2004).
Example 1
Artificially synthesizing interference sequences of mouse IL-33(250), IL-33(456) and IL-33(594), and adding PstI and BamHI enzyme cutting sites at two ends respectively to obtain the shIL-33(250) nucleotide sequence shown as SEQ ID NO 1; the nucleotide sequence of the shIL-33(456) is shown as SEQ ID NO. 2; the nucleotide sequence of shIL-33(594) is shown as SEQ ID NO. 3.
Example 2
Carrying out double enzyme digestion on the pDC316-ZsGreen1-shRNA vector by using restriction endonucleases PstI and BamHI, carrying out incubation for 4h at constant temperature of 37 ℃ under the enzyme digestion reaction condition, detecting the size of a band cut by enzyme digestion by using 1% agarose gel electrophoresis of an enzyme digestion product as shown in Table 1, and recovering a linear vector to obtain the linearized pDC316-ZsGreen1-shRNA vector. The results are shown in FIG. 1.
TABLE 1 enzyme digestion System
| Reagent | Volume (μ L) |
| CutSmart Buffer(10×) | 5 |
| |
|
| Endonuclease BamHI | |
| 2 | |
| pDC316-ZsGreen1-shRNA plasmid | 10μg |
| ddH2O | Make up to 50. mu.L |
The three artificially synthesized IL-33 interference fragments prepared in example 1 were connected with linearized vectors by T4, the connection system is shown in Table 2, the connection reaction condition is 16 ℃ for overnight connection, and three pDC316-ZsGreen1-shIL33 adenovirus expression plasmids are respectively marked as pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456), and pDC316-ZsGreen1-shIL-33 (594).
TABLE 2 connection System
The three pDC316-ZsGreen1-shIL33 adenovirus expression plasmids obtained were subjected to single enzyme digestion verification with the restriction enzyme BamHI, respectively, the single enzyme digestion system is shown in Table 3, and the reaction conditions were incubation at 37 ℃ for 4 h. And (5) verifying by agarose gel electrophoresis, wherein the positive clone is obtained if the clone is not cut. And meanwhile, BamHI and PstI double enzyme digestion verification is adopted, and the verification method is the same as single enzyme digestion verification. The results are shown in FIG. 2.
TABLE 3 Single enzyme digestion System
| Reagent | Volume (μ L) |
| CutSmart Buffer(10×) | 5 |
| |
2 |
| pDC316-ZsGreen1-shIL33 | 10μg |
| ddH2O | Make up to 50. mu.L |
Example 3
A shRNA recombinant adenovirus packaging method for interfering a mouse IL-33 gene comprises the step of co-transfecting HEK293 cells with three recombinant plasmids pDC316-ZsGreen1-shIL-33 and a shuttle plasmid pBHGlox respectively to realize the packaging of recombinant adenovirus. The preparation method specifically comprises the following steps:
1) HEK293 cells were seeded in 6-well plates to give approximately 5X 10 cells per well5A plurality of;
2) the three recombinant adenovirus plasmids pDC316-ZsGreen1-shIL-33 and the shuttle plasmid pBHGlox (delata) E1,3Cre prepared in example 2 were respectively adjusted to have a total mass of 4 μ g, and prepared into a mixed solution Mix1 with 500 μ L of DMEM medium;
3) preparing 12 mu L of liposome Lipofectamine TM2000 and 500 mu L of DMEM culture medium into mixed solution Mix2, uniformly mixing Mix1 and Mix2, and standing for 10 minutes;
4) dropwise adding the mixed solution obtained in the step 3) after standing into a 6-hole plate, uniformly mixing in a shape of a Chinese character '8', and placing at 37 ℃ for 5% CO2Culturing in an incubator, and changing a DMEM medium containing 10% fetal calf serum after 12 h.
5) After 48h, fluorescence was observed, half the fluid change was performed after 5d, and the virus was collected after 10 d.
6) And (2) repeatedly freezing and thawing the cells and the culture medium for 3 times, collecting the crushed cells and the culture medium, centrifuging at 3000rpm for 10min, and collecting the supernatant to obtain three shRNA recombinant adenoviruses interfering the mouse IL-33 gene, which are marked as recombinant adenovirus shIL-33(250), recombinant adenovirus shIL-33(456) and recombinant adenovirus shIL-33 (594).
After the three recombinant plasmids pDC316-ZsGreen1-shIL-33 infect the HEK293 cells, the infected cells can express green fluorescent protein (the result is shown in figure 3), and the results show that the three recombinant plasmids pDC316-ZsGreen1-shIL-33 successfully infect the HEK293 cells.
Example 4
Amplification and purification method of shRNA recombinant adenovirus
1) Inoculating HEK293 cells into a 10cm cell culture dish to ensure that the fusion degree reaches 70%;
2) replacing cell culture solution with serum-free DMEM medium, adding 1mL adenovirus stock solution, and standing at 37 deg.C with 5% CO2Incubating for 2h in an incubator;
3) and when the cells fall off by 50%, repeatedly freezing and thawing the cells and the culture medium for 3 times, collecting the broken cells and the culture medium, centrifuging at 3000rpm for 10min, collecting the supernatant, and repeating for multiple times to obtain the shRNA recombinant adenovirus interfering the IL-33 gene of the mouse.
Example 5
The specific implementation method of the titer determination method of shRNA recombinant adenovirus interfering mouse IL-33 gene comprises the following steps:
1) HEK293 cells were seeded in 96-well plates to give approximately 1X 10 cells per well4After being cultured in a 5% CO2 incubator at 37 ℃ for 24 hours, the cell culture medium is replaced by a DMEM medium containing 1% fetal calf serum;
2) the concentrated and purified recombinant adenovirus is diluted into ten groups in a gradient way (10)-1，10-2，10-3，10-4，10-5，10-6，10-7，10-8，10-9，10-10) Each group was repeated 8 times, and each was added dropwise to a 96-well plate and incubated at 37 ℃ with 5% CO2Culturing in an incubator;
3) and counting the number of fluorescence positive cells under a fluorescence microscope after 24h, wherein the calculation formula is as follows: virus titer (PFU/mL) ═ average number of fluorescence per well x 10)/dilution.
All three recombinant adenoviruses prepared above were concentrated to 7X 10 titres9(PFU/mL)。
Example 6
Three recombinant adenoviruses are uniformly mixed according to the volume ratio of 1:1:1, the tail vein is injected into a mouse, the mouse is dissected after 24 hours, and the liver is taken out for Western Blot and immunohistochemical detection.
Western Blot：
1. 10% separation gel and 5% concentration gel, and electrophoresis solution and membrane transfer solution were prepared (formula is referred to "molecular cloning experimental manual" (fourth edition)). And (3) well mixing the electrophoresis solution, placing the glue-making plate and the glue-connecting assembly in an electrophoresis device, pouring the electrophoresis solution to ensure no leakage, slightly pulling out the comb, adding samples, carrying out electrophoresis for 2 hours at a voltage of 120V, wherein the protein content of each hole is 10 mu g. And (3) mixing and uniformly mixing the film transfer liquid, and then placing the mixture in a refrigerator for precooling at 4 ℃.
2. Film transfer: cutting and separating a PVDF membrane with the size of 0.22 mu m, and activating in methanol for 5min in advance; after electrophoresis is finished, taking out the gel block, cutting off the concentrated gel and the redundant separation gel, and keeping the gel block, the filter paper and the sponge pad soaked in the precooled membrane transferring liquid; taking out the rotary film plate, placing the rotary film plate with the black surface facing downwards in sequence from bottom to top: sponge, filter paper, a rubber block, an activated PVDF membrane, filter paper and sponge, wherein bubbles remained in the middle are avoided in the process of the device, the device is placed in a membrane rotating groove after being filled, membrane rotating liquid is poured in, and an ice box is added; the constant current of 200mA flows through the membrane for 60 min.
3. Blocking and antibody incubation: preparing 5% skimmed milk as confining liquid with 1 × TBST, taking out the membrane after membrane transfer is finished, and sealing at room temperature for 2 h; after sealing, adding TBST to clean residual sealing liquid, cutting into strips of membranes with corresponding sizes, and respectively subpackaging in each primary antibody for incubation at 4 ℃ overnight; recovering primary antibody, washing the membrane with TBST for 5 times (5 min each time); adding the correspondingly diluted secondary antibody, and incubating for 1h at room temperature; the secondary antibody was recovered, the membrane was washed with TBST 5 times for 5min each, and the membrane was stored in TBST.
4. Chemiluminescence: preparing a developing solution: preparing and uniformly mixing the developing solution A and the developing solution B according to the ratio of 1: 1; and (3) developing: removing TBST on the membrane, uniformly covering the membrane with the developing solution, and developing for 5min in a dark place; exposure: and the chemiluminescence imaging system performs luminescence imaging, and a picture is selected according to the exposure time.
Immunohistochemistry:
1. antigen retrieval: paraffin section dewaxing, PBS washing 3 times, circling the tissue on the glass slide by a organizing pen, acting 3% hydrogen peroxide for 10min, and PBS washing 3 times; 0.01M citrate (pH6.0) is repaired at high temperature to expose epitope, cooled to room temperature, and washed 3 times with PBS.
2. Blocking and antibody incubation: blocking the tissue sample with 5% BSA at 37 deg.C for 30 min; washing with PBS for 1 time, adding primary antibody, and standing overnight at 4 deg.C; PBS washing for 3 times; add secondary antibody drop wise, incubate 30min at 37 ℃ and wash 3 times with PBS.
3. Color development: according to A/B: preparing a streptavidin biotin marker (SABC) reagent with a ratio of DDW to 1:100, dripping the reagent on the tissue, and incubating for 30min at 37 ℃; washing with PBS for 3 times, preparing DBA developing solution, dripping the developing solution while observing under a mirror, and washing off the developing solution when brown marks are seen.
4. Nuclear staining: and (3) performing hematoxylin dip-dyeing for 3min, washing off the hematoxylin, drying in the air, and dripping neutral gum for sealing.
The detection result of Western Blot identification is shown in FIG. 4. Compared with the protein expression of an internal reference gene beta-Actin, the expression level of the mouse IL-33 protein in a mouse injected with shRNA recombinant adenovirus interfering the mouse IL-33 gene is obviously reduced.
The immunohistochemical identification results are shown in FIG. 5. As can be seen from fig. 5, compared with the control group in which mature liver parenchymal cells can normally express IL-33, the expression level of the IL-33 protein of parenchymal cells in the adenovirus group is significantly reduced, which indicates that the shRNA recombinant adenovirus provided by the present invention is qualified in interference efficiency.
Comparative example 1
Separately injecting three recombinant adenoviruses prepared in example 5 into mice separately in tail vein, performing 24h later autopsy, taking liver, extracting liver RNA by TRIzol method, removing DNA in RNA sample, and performing reverse transcription, wherein the specific steps refer to molecular cloning experimental guidance (fourth edition).
The relative expression level of IL-33 was determined by fluorescent quantitative PCR using the cDNA obtained by reverse transcription.
The fluorescent quantitative PCR reaction system is shown in Table 4.
TABLE 4 fluorescent quantitative PCR reaction system reagent
Fluorescent quantitative PCR reaction program:
amplification: controlling the temperature at 50 ℃ for 2min, pre-denaturing at 95 ℃ for 2min, denaturing at 95 ℃ for 30s, and annealing at 60 ℃ for 30s, wherein the total number is 40 cycles; dissolution curve: 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15 s.
The fluorescent quantitation primer sequences are shown in Table 5.
TABLE 5 fluorescent quantitation primer sequences
The results of fluorescence quantification are shown in FIG. 6, and the interference effect is weaker than that of the three recombinant adenoviruses when the recombinant adenovirus is used alone.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yangzhou university
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Claims (10)
1. A group of shRNAs for targeting and interfering with IL-33 gene or protein expression, which is characterized by comprising shIL-33(250), shIL-33(456) and shIL-33 (594);
the nucleotide sequence of the shIL-33(250) is shown as SEQ ID NO:1 is shown in the specification;
the nucleotide sequence of the shIL-33(456) is shown in SEQ ID NO:2 is shown in the specification;
the nucleotide sequence of the shIL-33(594) is shown in SEQ ID NO:3, respectively.
2. A group of recombinant adenovirus vectors for targeting and interfering IL-33 gene or protein expression, which is characterized by comprising shRNA of claim 1.
3. The recombinant adenovirus vector according to claim 2, wherein the shRNA comprises shIL-33(250), shIL-33(456) and shIL-33(594) which are respectively inserted into a polyclonal site of the pDC316-ZsGreen1-shRNA adenovirus vector.
4. The recombinant adenovirus vector according to claim 3, wherein the multiple cloning site is PstI and BamHI cleavage site.
5. The method for constructing a recombinant adenovirus vector according to any one of claims 2 to 4, comprising the steps of:
1) adding PstI and BamHI cleavage sites to both ends of the shIL-33(250), shIL-33(456) or shIL-33(594) in the shRNA of claim 1, and removing the first G base of the BamHI cleavage site to obtain the shIL-33(250), shIL-33(456) or shIL-33(594) having cleavage sites at both ends;
2) carrying out double enzyme digestion on the adenovirus vector pDC316-ZsGreen1-shRNA by using restriction endonucleases PstI and BamHI to obtain a linearized pDC316-ZsGreen1 vector;
3) carrying out T4 connection on the linearized pDC316-ZsGreen1 vector obtained in the step 2) and the shIL-33(250), shIL-33(456) or shIL-33(594) with the enzyme cutting sites at two ends obtained in the step 1) to obtain three recombinant adenovirus vectors which are respectively named as: pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456), pDC316-ZsGreen1-shIL-33 (594);
there is no chronological restriction between step 1) and step 2).
6. The construction method according to claim 5, wherein T4 is ligated and then BamHI is used to verify three recombinant adenovirus expression plasmids.
7. A method for packaging a recombinant adenovirus, comprising the steps of:
A. pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) obtained by the construction method of claim 5 or 6 and a shuttle plasmid pBHGlox (delta) E1,3Cre are mixed according to the proportion of 5-6: 3-4, and mixing with a DMEM culture medium to prepare a mixed solution Mix 1;
B. preparing a mixed solution Mix2 by using liposome Lipofectamine TM2000 and DMEM medium;
C. uniformly mixing the Mix1 and Mix2, and standing to obtain an invasion liquid;
D. c, infecting the eukaryotic cells with the infection liquid obtained in the step C, observing fluorescence after 48h, and collecting viruses after 10 days to obtain recombinant adenovirus shIL-33(250), recombinant adenovirus shIL-33(456) and recombinant adenovirus shIL-33 (594);
there is no chronological restriction between step a and step B.
8. The packaging process of claim 7, wherein the total mass concentration of pDC316-ZsGreen1-shIL-33(250), pDC316-ZsGreen1-shIL-33(456) or pDC316-ZsGreen1-shIL-33(594) and shuttle plasmid pbhglox (delta) E1,3Cre in Mix1 is 2 μ g/mL.
9. The recombinant adenovirus prepared by the recombinant adenovirus packaging method according to claim 7 or 8, wherein each recombinant adenovirus comprises shRNA for the targeted interference of IL-33 gene or protein expression according to claim 1.
10. Use of the shRNA according to claim 1, the recombinant adenovirus vector according to any one of claims 2 to 4 or the recombinant adenovirus according to claim 9 in the preparation of a medicament for treating hepatic fibrosis injury.
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