CN110964727A - shRNA lentiviral expression vector construction method for specifically inhibiting c-myc gene expression and application thereof - Google Patents
shRNA lentiviral expression vector construction method for specifically inhibiting c-myc gene expression and application thereof Download PDFInfo
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Abstract
The invention relates to a construction method and application of shRNA (short hairpin ribonucleic acid) lentiviral expression vector for specifically inhibiting c-myc gene expression. The shRNA for specifically inhibiting the expression of the c-myc gene comprises a target sequence, a stem-loop structure, a complementary sequence of the target sequence and a termination site which are sequentially connected, wherein the target sequence is shown as SEQ ID No.1, or the target sequence is shown as SEQ ID No.2, or the target sequence is shown as SEQ ID No. 3. The shRNA can continuously, stably, efficiently and specifically inhibit the expression of the c-myc gene in human cells.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a shRNA lentiviral expression vector for specifically inhibiting c-myc gene expression, and a construction method and application thereof.
Background
The c-myc gene is one of the important members of myc gene family, is a translocatable gene, is a regulatable gene regulated by various substances, and is a gene capable of realizing unlimited proliferation of cells, obtaining an immortalization function and promoting cell division. The c-myc gene is involved in cell withering, and is involved in the development of various tumors.
Expression of the c-myc gene is generally associated with the growth state of the cell. For example, stimulation of fibroblasts by growth factors may lead to enhanced expression of c-myc; in contrast, c-myc expression is reduced upon cell differentiation, and studies with c-myc expression constructs or antisense oligodeoxynucleotides during cell culture have shown that c-myc also plays a role during the G0 to S phase of cells, indicating that changes in c-myc expression are associated with the proliferative and differentiative state of cells, and that their expression products play a role in regulating cell growth, differentiation or malignant transformation.
In the prior art, MCF7 cells are transfected after shRNA is connected to a vector pEGFPC1/U6 to inhibit the expression of c-myc genes in MCF7 cells, but the interference effect of the transient transfection method is short and unstable.
Disclosure of Invention
In view of this, it is necessary to provide an shRNA which has a significant and specific effect of inhibiting the expression of the c-myc gene and which is stable in effect.
An shRNA for specifically inhibiting c-myc gene expression comprises a target sequence, a stem-loop structure, a complementary sequence of the target sequence and a termination site which are connected in sequence, wherein,
the target sequence is shown in SEQ ID NO.1, or,
the target sequence is shown in SEQ ID NO.2, or,
the target sequence is shown in SEQ ID No. 3.
An shRNA lentiviral expression vector for specifically inhibiting c-myc gene expression comprises a vector and the shRNA inserted in the vector.
In one embodiment, the shRNA inserted into the vector is a double-stranded nucleotide fragment, and two strands of the double-stranded nucleotide fragment are respectively shown as SEQ ID No. 4-SEQ ID No. 5; or two chains of the double-stranded nucleotide fragment are respectively shown as SEQ ID No. 6-SEQ ID No. 7; or two chains of the double-stranded nucleotide fragment are respectively shown as SEQ ID No. 8-SEQ ID No. 9.
In one embodiment, the vector is a lentiviral expression vector.
In one embodiment, the vector is pLVX-shRNA1, the vector comprises a BamHI enzyme cutting site, an EcoRI enzyme cutting site and a KpnI enzyme cutting site, the shRNA comprises a BamHI enzyme cutting site cohesive end and an EcoRI enzyme cutting site cohesive end, and the shRNA is inserted between the BamHI enzyme cutting site and the EcoRI enzyme cutting site in a forward direction.
A method for constructing shRNA lentiviral expression vector for specifically inhibiting c-myc gene expression comprises the following steps:
providing a double-stranded nucleotide fragment, wherein the double-stranded nucleotide fragment comprises the shRNA for specifically inhibiting the expression of the c-myc gene; and
and inserting the double-stranded nucleotide fragment into a lentiviral expression vector to obtain the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
In one embodiment, the step of inserting the double-stranded nucleotide fragment into a lentiviral expression vector is followed by the step of transferring the lentiviral expression vector containing the double-stranded nucleotide fragment into a host cell.
In one embodiment, the host cell is a 293FT cell.
The recombinant engineering bacterium contains at least one of the shRNA for specifically inhibiting the expression of the c-myc gene and the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
The shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene or the application of the recombinant engineering bacteria in preparing medicines for treating diseases related to abnormal expression of the c-myc gene.
A pharmaceutical composition comprises the shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene or the recombinant engineering bacteria.
The research has conducted a great deal of research on the aspect of specific inhibition of c-myc gene expression, and unexpectedly found that the shRNA can continuously, stably and efficiently inhibit the c-myc gene expression by specifically combining with a part of sequences in the c-myc gene. Experiments prove that the shRNA lentiviral expression vector containing the shRNA can be transfected into human cells, and the transfection efficiency is high. The expression of the c-myc gene in the hepatocyte L02 after the c-myc gene is silenced is obviously inhibited, and the gene expression quantity is reduced by 81 percent compared with that of the hepatocyte L02; after the c-myc gene is silenced, the expression of the c-myc protein of the liver cell L02 is reduced by 70 percent. The c-myc gene expression in the hepatocytes L02 of the control group had little effect; and after the hepatocyte L02 after c-myc gene silencing is continuously subcultured for 20 generations, collecting and respectively detecting the expression quantity of the c-myc gene and the protein, wherein the expression of the c-myc gene in the hepatocyte L02 after the c-myc gene silencing is consistent with the initial detection result, which shows that the shRNA for inhibiting the expression of the c-myc gene can continuously, stably, efficiently and specifically inhibit the expression of the c-myc gene in the human source cell.
Drawings
FIG. 1 is a diagram of the double-restriction electrophoresis of the lentiviral expression vector of example 1;
FIG. 2 is a partial sequencing result of the pLVX-c-myc-shRNA1 lentiviral expression vector in example 1;
FIG. 3 is a partial sequencing result of the pLVX-c-myc-shRNA2 lentiviral expression vector in example 1;
FIG. 4 is a partial sequencing result of the pLVX-c-myc-shRNA3 lentiviral expression vector of example 1;
FIG. 5 is a partial sequencing result diagram of the pLVX-shRNAC lentiviral expression vector of example 1;
FIG. 6 shows the mRNA expression of c-myc in example 2 after the hepatocyte L02 was transfected with four viruses respectively;
FIG. 7 is the qPCR results of example 3;
FIG. 8 is a diagram showing the result of the Western blot detection electrophoresis in example 3;
FIG. 9 is a graph showing the quantitative results of Western blot detection in example 3;
FIGS. 10 to 16 are graphs showing the effect of PM2.5 on the expression of c-fos, c-myc, k-ras, p53, Caspase-3, Caspase-8 and Caspase-9 in hepatocytes after silencing of L02 and c-myc genes in hepatocytes.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The shRNA for specifically inhibiting the expression of the c-myc gene comprises a target sequence, a stem-loop structure, a complementary sequence of the target sequence and a termination site which are connected in sequence. The complement of the target sequence of the shRNA that specifically inhibits expression of the c-myc gene can target a specific sequence of the c-myc gene such that expression of the c-myc gene is inhibited.
In one embodiment, the target sequence is as set forth in seq.id No. 1.
In one embodiment, the target sequence is as set forth in seq.id No. 2.
In one embodiment, the target sequence is as set forth in seq.id No. 3.
The stem-loop structure is used for enabling the shRNA to be in a stem loop. Further, the base sequence of the stem-loop structure is 5 '-TTCAAGAGAGA-3'.
The termination site is the transcription termination site of RNA polymerase. In one embodiment, the termination site is the transcription termination site of rnapol III polymerase. Further, the base sequence of the termination site was 5 '-TTTTT-3'.
In one embodiment, the shRNA further comprises a cohesive end of the cleavage site corresponding to the vector, enabling the shRNA to be ligated to the cleaved vector. Further, the shRNA comprises a cohesive end of a BamHI enzyme cutting site, a target sequence, a stem-loop structure, a complementary sequence of the target sequence, a termination site and a cohesive end of an EcoRI enzyme cutting site which are connected in sequence from the 5 'end to the 3' end.
Of course, the cohesive ends of the cleavage sites in the shRNA are not limited to the cohesive ends of the BamHI cleavage site and the cohesive ends of the EcoRI cleavage site, and may be other cohesive ends of the cleavage sites as long as the shRNA can be ligated to a vector to construct a lentiviral expression vector.
In one embodiment, the shRNA is present as a double-stranded nucleotide fragment. shRNA in the form of a double-stranded nucleotide fragment can be inserted into a viral vector to transfect a host cell, so as to continuously, stably, efficiently and specifically inhibit the expression of a c-myc gene in the host cell.
Furthermore, two strands of the shRNA are respectively shown as SEQ ID No. 4-SEQ ID No. 5. Wherein, the sense strand is shown as SEQ ID No.4, and the antisense strand is shown as SEQ ID No. 5.
Furthermore, two strands of the shRNA are respectively shown as SEQ ID No. 6-SEQ ID No. 7. Wherein, the sense strand is shown as SEQ ID No.6, and the antisense strand is shown as SEQ ID No. 7.
Furthermore, two strands of the shRNA are respectively shown as SEQ ID No. 8-SEQ ID No. 9. Wherein, the sense strand is shown as SEQ ID No.8, and the antisense strand is shown as SEQ ID No. 9.
The research has conducted a great deal of research on the inhibition of the expression of the c-myc gene, and unexpectedly found that the shRNA can continuously, stably and efficiently inhibit the expression of the c-myc gene by specifically binding with a part of sequences in the c-myc gene. Experiments prove that the shRNA lentiviral expression vector containing the shRNA can be transfected into human cells, and the transfection efficiency is high. The expression of the c-myc gene in the hepatocyte L02 after the c-myc gene is silenced is obviously inhibited, and the gene expression quantity is reduced by 81 percent compared with that of the hepatocyte L02; after the c-myc gene is silenced, the expression of the c-myc protein of the liver cell L02 is reduced by 70 percent. And the c-myc gene expression in the liver cell L02 of the control group has almost no influence, which shows that the shRNA for specifically inhibiting the c-myc gene expression can continuously, stably, efficiently and specifically inhibit the c-myc gene expression in the human source cell.
An shRNA slow virus expression vector for specifically inhibiting c-myc gene expression comprises a vector and the shRNA inserted into the vector.
Specifically, the vector includes a base sequence, a resistance gene sequence, a multiple cloning site sequence, and a promoter sequence.
In one embodiment, the resistance gene sequence comprises an ampicillin resistance gene sequence.
The multiple cloning site sequence contains multiple restriction endonuclease single recognition sites, and can provide multiple insertable positions or insertion schemes for foreign DNA.
In one embodiment, the vector is a lentiviral expression vector. Further, the lentiviral expression vector was pLVX-shRNA 1. The pLVX-shRNA1 is selected because the pLVX-shRNA1 vector carries a puromycin resistance screening label, can be used for screening cell strains, and can quickly and efficiently complete cell strain identification.
The pLVX-shRNA1 comprises a BamHI restriction site, an EcoRI restriction site and a KpnI restriction site. The BamHI enzyme cutting site of pLVX-shRNA1 corresponds to the enzyme cutting site of shRNA, and the EcoRI enzyme cutting site of pLVX-shRNA1 corresponds to the EcoRI enzyme cutting site of shRNA, so that corresponding cohesive ends are formed after BamHI and EcoRI enzyme cutting, and the enzyme-cut vector and the enzyme-cut shRNA form an shRNA lentiviral expression vector for specifically inhibiting c-myc gene expression.
The KpnI cleavage site of pLVX-shRNA1 can be matched with the BamHI cleavage site thereof to verify whether the shRNA is successfully inserted into pLVX-shRNA 1.
In one embodiment, the shRNA is inserted forward between the BamHI cleavage site and the EcoRI cleavage site of the lentiviral expression vector.
This study conducted extensive research on the selection of target sequences for inhibiting c-myc gene expression, the selection of shRNA, the selection of vectors, and the insertion site of shRNA, and unexpectedly found that inserting the shRNA into a lentiviral expression vector successfully constructed a lentiviral expression vector capable of specifically inhibiting c-myc gene. The transfection efficiency of the lentivirus expression vector into the human cells is high, the dosage of the expression vector is small, and the lentivirus expression vector can continuously, stably and efficiently inhibit the expression of the c-myc gene.
The method for constructing the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene comprises the following steps:
s110, providing any shRNA for specifically inhibiting the expression of the c-myc gene.
Specifically, the sense strand and the antisense strand of the shRNA are obtained by means of gene synthesis. The sense and antisense strands are then mixed and annealed to form the shRNA.
S120, inserting the shRNA into a lentiviral expression vector to obtain the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
Specifically, the shRNA is inserted into a lentiviral expression vector subjected to double digestion treatment by a restriction enzyme BamHI and a restriction enzyme EcoRI, so as to obtain the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
Further, the lentiviral expression vector is subjected to double enzyme digestion treatment by restriction enzyme BamHI and restriction enzyme EcoRI, a gap is opened, and the cohesive end of the enzyme digestion site of the lentiviral expression vector is formed, so that the shRNA for specifically inhibiting the expression of the c-myc gene is connected with the cohesive end of the enzyme digestion site of the lentiviral expression vector, and the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene is obtained.
Specifically, after the vector is subjected to double digestion by using a restriction enzyme BamHI and a restriction enzyme EcoRI, the shRNA is connected between the BamHI digestion site and the EcoRI digestion site of the double digestion treated pLVX-shRNA1 by using DNA ligase, and the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene is obtained. Further, the lentiviral expression vector was pLVX-shRNA 1. The DNA ligase is T4 DNA ligase.
In one embodiment, after the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene is obtained, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene is amplified and identified.
The construction method of the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene is simple in operation, and the shRNA lentiviral expression vector capable of specifically inhibiting the c-myc gene is successfully constructed by inserting the shRNA containing the target sequence into the lentiviral expression vector. Experimental results show that the lentivirus expression vector can be transfected into human cells, the transfection efficiency is high, the dosage of the expression vector is small, and the expression of c-myc genes in the human cells can be continuously, stably, efficiently and specifically inhibited.
One embodiment of a lentivirus that specifically inhibits the expression of a c-myc gene is made by: cotransfecting the shRNA slow virus expression vector for specifically inhibiting the expression of the c-myc gene and a packaging vector into a host cell to obtain more shRNA slow virus expression vectors for specifically inhibiting the expression of the c-myc gene. Specifically, the host cells are 293FT cells. Mixing the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene with a packaging vector, co-transfecting the shRNA lentiviral expression vector into 293FT cells, culturing the 293FT cells after transfection, collecting supernatant, and filtering to obtain virus supernatant containing the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
Furthermore, the time for culturing the 293FT cells after transfection is 24-48 h. Virus titers were detected using the Lenti-X GoStix kit.
The packaging vector may be any other vector as long as it can promote amplification and secretion of a lentiviral expression vector that specifically inhibits the expression of the c-myc gene. Of course, the packaging vector may be omitted, so long as the lentiviral expression vector itself is capable of amplification and secretion.
The shRNA lentivirus for specifically inhibiting the expression of the c-myc gene can be transduced into a human source cell, and the lentivirus can continuously, stably and efficiently inhibit the expression of the c-myc gene in the human source cell.
One embodiment of a cell in which expression of a c-myc gene is inhibited is prepared by: the hepatocyte L02 infected by the virus containing the shRNA lentivirus expression vector for specifically inhibiting the expression of the c-myc gene is used, and the infected hepatocyte L02 is cultured to obtain the cell with the expression of the c-myc gene inhibited.
Specifically, the hepatocyte L02 is cultured until the cell confluency reaches 60% -90%, the hepatocyte L02 is infected by the virus containing the lentivirus expression vector for specifically inhibiting the expression of the c-myc gene, and the hepatocyte is cultured for 24-48 hours at 36.5-38 ℃ to obtain the cell with the expression of the c-myc gene inhibited.
The recombinant engineered bacterium of an embodiment contains at least one of the shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene, and the lentivirus for specifically inhibiting the expression of the c-myc gene.
The recombinant engineering bacteria contain at least one of the shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene and the lentivirus for specifically inhibiting the expression of the c-myc gene, and can continuously, stably and efficiently inhibit the expression of the c-myc gene.
The shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentivirus expression vector for specifically inhibiting the expression of the c-myc gene, the lentivirus for specifically inhibiting the expression of the c-myc gene or the recombinant engineering bacteria are applied to the preparation of medicines for treating diseases related to abnormal expression of the c-myc gene.
The pharmaceutical composition of one embodiment can be used for treating diseases associated with abnormal expression of the c-myc gene, in particular cancer caused by PM 2.5. The pharmaceutical composition comprises the shRNA for specifically inhibiting the expression of the c-myc gene, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene, the lentivirus for specifically inhibiting the expression of the c-myc gene or the recombinant engineering bacteria.
It will be appreciated that pharmaceutically acceptable adjuvants may also be included in the pharmaceutical composition.
The following are specific examples
In the following examples, unless otherwise specified, the experimental procedures without specifying the specific conditions are generally carried out according to conventional conditions, for example, the conditions described in the molecular cloning's Experimental guidelines [ M ] (Beijing: scientific Press, 1992) by Sammbruke, EF Friech, T Mannich, et al (decoded by gold winter goose, Rimeng maple, et al) or the procedures recommended by the manufacturers of the kits. The reagents used in the examples are all commercially available.
Example 1
Construction of expression vector for specifically inhibiting c-myc gene expression
(1) Preparation of shRNA
Two strands of four shRNAs were synthesized by Compton Biotechnology engineering (Shanghai) Inc. Four shrnas that specifically interfere with c-myc: c-myc-1, c-myc-2, c-myc-3 and shRNAC, wherein the shRNAC is a control group, and the c-myc-1 contains a target sequence shown as SEQ ID No. 1; c-myc-2 is a target sequence shown as SEQ ID No. 2; c-myc-3 contains a target sequence shown as SEQ ID No. 3; the shRNAC contains a target sequence shown as SEQ ID No. 10. Wherein, the sense strand of c-myc-1 (c-myc-T1) is shown as SEQ ID No.4, the antisense strand of c-myc-1 (c-myc-D1) is shown as SEQ ID No.5, the sense strand of c-myc-2 (c-myc-T2) is shown as SEQ ID No.6, the antisense strand of c-myc-2 (c-myc-D2) is shown as SEQ ID No.7, the sense strand of c-myc-3 (c-myc-T3) is shown as SEQ ID No.8, the antisense strand of c-myc-3 (c-myc-D3) is shown as SEQ ID No.9, the sense strand of shrNAC (shrNAC-TC) is shown as SEQ ID No.11, and the antisense strand of shrNAC (shrnNAC-DC) is shown as SEQ ID No. 12. The specific sequence is shown in Table 1.
TABLE 1
(2) Construction of lentivirus expression vector for specifically inhibiting c-myc gene expression
The pLVX-shRNA1 was digested simultaneously with EcoR1 and BamH1 at 37 ℃ for 30min, and then the restriction enzyme was added again to the mixture and digested at 37 ℃ for 30 min. The enzyme digestion reaction system is as follows:
TABLE 2
The digested vector was recovered by agarose Gel electrophoresis, the procedure is described in the Gel extraction kit (D2500-01) of OMEGA.
(3) Annealing of c-myc-shRNA
The double strands of the c-myc-shRNA were mixed uniformly as in Table 3, then placed in boiling water and allowed to cool naturally to room temperature (26 ℃ C.) and annealing was completed. Then adding 1mLddH into the c-myc-shRNA after the completion of annealing2O。
TABLE 3
(4) Construction of recombinant interference vectors
And respectively connecting the c-myc-1 fragment, the c-myc-2 fragment, the c-myc-3 fragment and the shRNAC fragment with the cohesive ends, which are formed after annealing, with the cut pLVX-shRNA 1. The ligation reaction system is shown in Table 4, and ligation is carried out overnight at 16 ℃ to obtain ligation products corresponding to c-myc-1, c-myc-2, c-myc-3 and shRNAC.
TABLE 4
(5) Transformation of
mu.L of each ligation product was transformed into 100. mu.L of JM107 competent cells: and uniformly mixing the ligation product and competent cells, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 70s, immediately placing on ice for 2min, adding 150 mu L of TB culture medium preheated to room temperature, carrying out shake culture at constant temperature of 37 ℃ for 1h at 250rpm, taking 100 mu L of TB culture medium by using a pipette, uniformly coating the TB culture medium on a 100 mu g/mL Ampicillin-resistant TB plate, inverting, and carrying out culture in a constant temperature incubator at 37 ℃ overnight.
(6) Positive clone plasmid extraction and enzyme digestion identification
A single clone on a TB plate is picked up, added into a TB culture medium for culturing, a recombinant plasmid is extracted by using a plasmid miniprep kit, and then the plasmids KpnI + BamHI and KpnI + EcoRI are subjected to enzyme digestion and identification, and the result is shown in figure 1.
In FIG. 1, the marker lane corresponds to DNA marker; lane c-myc-1(K + B) corresponds to the ligation product of pLVX-shRNA1 with c-myc-1, transformed and then double digested with BamH1, KpnI; lane c-myc-1(K + E) corresponds to the product of the ligation of pLVX-shRNA1 with c-myc-1, transformed and then double digested with EcoRI and KpnI; lane c-myc-2(K + B) corresponds to the product of the ligation of pLVX-shRNA1 with c-myc-2, transformed and then double digested with BamH1, KpnI; lane c-myc-2(K + E) corresponds to the product of the ligation of pLVX-shRNA1 with c-myc-2, transformed and then double digested with EcoRI and KpnI; lane c-myc-3(K + B) corresponds to the ligation product of pLVX-shRNA1 with c-myc-3, transformed and then double digested with BamH1, KpnI; lane c-myc-3(K + E) corresponds to the product of the ligation of pLVX-shRNA1 with c-myc-3, transformed and then double digested with EcoRI and KpnI; lane shRNAC (K + B) corresponds to the product of the ligation product of pLVX-shRNA1 and shRNAC after transformation and double digestion with BamH1 and KpnI; lane shRNAC (K + E) corresponds to the product of the ligation of pLVX-shRNA1 with shRNAC, transformed, and then EcoRI, KpnI double-digested.
pLVX-shRNA1 has a BamH1 restriction site, a KpnI restriction site and an EcoRI restriction site, but after the EcoRI restriction site is bound to the cohesive end of EcoRI of the shRNA to form a lentiviral expression vector, the EcoRI restriction site cannot be cut again by EcoRI enzyme. Therefore, when the lentivirus expression vector is cut by EcoRI and KpnI, only one cutting site of KpnI is cut, and only one band with the molecular weight of more than 5000bp appears during electrophoresis. Thus, it was preliminarily determined from FIG. 1 that c-myc-1, c-myc-2, c-myc-3 and shRNAC having cohesive ends were successfully inserted into pLVX-shRNA 1.
(7) Sequencing
And (3) selecting bacteria of the positive clones after enzyme digestion identification for culturing, and performing sequencing identification on the bacterial liquid after culture, wherein sequencing is performed by Jinweizhi company, and the sequencing identification result is shown in figures 2-5. FIG. 2 is a partial sequence diagram of a lentiviral expression vector consisting of c-myc-1 and pLVX-shRNA1 (i.e., pLVX-c-myc-shRNA1), wherein the sense strand of c-myc-1 is located at positions 55-112. FIG. 3 is a partial sequence diagram of a lentiviral expression vector consisting of c-myc-2 and pLVX-shRNA1 (i.e., pLVX-c-myc-shRNA2), wherein the sense strand of c-myc-2 is located at positions 49-106. FIG. 4 is a partial sequence diagram of a lentiviral expression vector consisting of c-myc-3 and pLVX-shRNA1 (i.e., pLVX-c-myc-shRNA3), wherein the sense strand of c-myc-3 is located at positions 49-106. FIG. 5 is a partial sequence chart of a lentiviral expression vector (i.e., pLVX-shRNAC) composed of shRNAC and pLVX-shRNA1, wherein the sense strand of shRNAC is located at positions 52-109. Thus, as is clear from FIGS. 2 to 5, c-myc-1, c-myc-2, c-myc-3 and shRNAC having cohesive ends were successfully inserted into pLVX-shRNA 1.
Example 2
Preparation of lentivirus for specifically inhibiting c-myc gene expression
(1) Packaging of lentivirus: according to Lenti-XTMAccording to the instructions of the Lentiviral Expression Systems kit, pLVX-C-myc-shRNA1, pLVX-C-myc-shRNA2, pLVX-C-myc-shRNA3 and pLVX-shRNAC are respectively packaged with lentiviruses to obtain corresponding C-myc-shRNA1 virus, C-myc-shRNA2 virus, C-myc-shRNA3 virus and C-myc-C virus. The specific operation is as follows:
a. at 37 deg.C, 5% CO2293T cells were cultured in large numbers under the conditions and the digested cells were plated evenly.
b. When the fusion rate of the cells was 90%, transfection was prepared. Fully and uniformly mixing the transfection reagent, the packaging plasmid,
After the objective plasmid was obtained, the plasmid was dropped into a petri dish for transfection.
(2) Concentrating and purifying the lentiviruses obtained in the step (1), wherein the concentration and purification of each lentivirus are as follows: centrifuging the virus at 10000RPM and 4 ℃ for 10min, and collecting the virus supernatant. Placing the virus supernatant in an ultracentrifuge centrifuge tube, balancing the centrifuge tube, centrifuging the virus at 50000RPM and 4 ℃ for 2h, removing the supernatant, retaining the precipitated virus, adding 1mL of PBS to dissolve the virus, and filtering the dissolved virus by using a 0.22 mu m filter. Finally, the filtered virus is tested for titer and packaged.
(3) Determining the titer of each lentivirus obtained in the step (2) by adopting an RT-qPCR method, specifically: a. extracting lentivirus RNA: 100 μ L of C-myc-shRNA1 virus, C-myc-shRNA2 virus, C-myc-shRNA3 virus, C-myc-C virus and LV-GFP (Standard 10)7TU, fluorescently labeled lentivirus), total RNA was extracted using trizol, methods referenced to trizol instructions. Finally, the RNA was dissolved in 50. mu.L of DEHP water.
b. Reverse transcription: reverse transcription of RNA into cDNA Using reverse transcription kit (PrimeScript)TMRT reagentKit) were used for cDNA reverse transcription synthesis, on ice to formulate the reverse transcription system (10 μ L) shown in table 5:
TABLE 5
Reaction conditions are as follows: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ and infinity. After the reverse transcription was complete, 90. mu.L of RNase freedH2O was added
The cDNA was diluted and stored at-20 ℃.
c. And (3) fluorescent quantitative detection: and (3) detecting the Ct value of each group of lentiviruses by using the LV-GFP lentivirus cDNA as a standard substance through qPCR. Wherein the qPCR reaction system is shown in table 6 below.
TABLE 6
Wherein, the nucleotide sequence of the PCR Forward primer in the table 6 is as follows: 5'-TGTTGGGCACTGACAATTC-3' (SEQ ID No. 29); the nucleotide sequence of the PCR Reverse primer is as follows: 5'-CCGAAGGGACGTAGCAGAAG-3' (SEQ ID No. 30).
The reaction to establish the melting curve is continued after the standard curve analysis: melting experiments were carried out in the interval 55 ℃ to 95 ℃: reaction conditions are as follows: 95 ℃ 30s, 1 cycle, 55 ℃ 30s 40 cycles, 95 ℃ 5s, 60 ℃ 1min, 95 ℃ 15s, standard curve, melting curve and relative quantification. After the reaction is finished, a standard curve, a melting curve and a relative quantitative value are obtained.
Titers of each virus were calculated from the standard curve. C-myc-shRNA1 virus, C-myc-shRNA2 virus, C-myc-shRNA3 virus and C-myc-CThe virus is 5.23X 10 respectively7TU、4.96×107TU、6.04×107TU、5.51×107TU。
(4) Detecting the interference effect of each lentivirus obtained in the step (2):
a. inoculation of hepatocytes in L02 six well plates, 3X 105After culturing for 18h, the cell fusion degree reaches about 50% per well, and 10. mu.L (1X 10) is added7TU) virus supernatant.
b. The cells were cultured for an additional 24h, then the lentiviral-containing medium was removed and fresh RPIM1640 complete medium was added for an additional 24 h.
c. RNA was extracted from each group of cells and reverse transcribed into cDNA.
d. And using cDNA as a template, and detecting the relative expression quantity of the c-myc gene by fluorescence quantitative PCR. The reaction system of the fluorescent quantitative PCR is shown in Table 7. In Table 7, the sequence of the PCR Forward primer for the c-myc gene is 5'-CCTGGTGCTCCATGAGGAGA-3' (SEQ ID No. 13); the sequence of the PCR Reverse primer for the c-myc gene was 5'-TCCAGCAGAAGGTGATCCAGAC-3' (SEQ ID No. 14). Reaction conditions of fluorescent quantitative PCR: melting experiments were carried out in the range of 55 ℃ to 95 ℃.30 s at 95 ℃, 1 cycle, 30s at 55 ℃, 40 cycles, 5s at 95 ℃, 1min at 60 ℃, 15s at 95 ℃,
TABLE 7
e. After the reaction is finished, a standard curve, a melting curve and a relative quantitative value are obtained. And analyzing the relative expression quantity of the c-myc gene of each group of cells according to the detected data. The results are shown in table 8 and fig. 6. Group "C" in FIG. 6 corresponds to C-myc-C virus; the "m 1" group corresponds to the c-myc-shRNA1 virus; the "m 2" group corresponds to the c-myc-shRNA2 virus; the "m 3" group corresponds to the c-myc-shRNA3 virus. The ordinate of FIG. 6 shows the relative expression level of the c-myc gene.
TABLE 8
The c-myc-shRNA1 virus, the c-myc-shRNA2 virus and the c-myc-shRNA3 virus can effectively and specifically inhibit the expression of the c-myc gene, wherein the interference effect of the c-myc-shRNA2 virus is optimal.
Example 3
construction and identification of c-myc gene silencing cell strain
(1) C-myc gene silencing cell strain constructed by using c-myc-shRNA2 virus; the specific operation is as follows:
a. inoculation of hepatocytes in L02 six well plates, 3X 105After culturing for 18h, the cell fusion degree reaches about 50% per well, and 10. mu.L (1X 10) is added7TU) was added to the virus supernatant with polybrene to a final concentration of 5. mu.g/mL and mixed well.
b. The cells were cultured for an additional 24h, then the lentiviral-containing medium was removed and fresh RPIM1640 complete medium was added for an additional 24 h.
c. 48h after addition of virus, 1. mu.g/mL Puromycin was added to the virus-infected cells. The liquid is changed every 2d, and the screening time is 7 d.
d. And after the screening is finished, removing the complete culture medium containing Puromycin in the culture flask, and adding a normal complete culture medium to enable the cells to normally grow to obtain the c-myc gene silencing cell strain.
(2) And (3) identifying the c-myc gene silencing cell strain obtained in the step (1) by using real-time fluorescent quantitative PCR (qPCR):
1) and (3) respectively inoculating the liver cell L02 and the c-myc gene silencing cell obtained in the step (1) into a 6-well plate. When the cell density reaches 80-90%, extracting the total RNA of each group of cells by using an RNeasy Mini Kit, and performing reverse transcription on the mRNA into cDNA by using a PrimeScript RTreagent Kit; reverse transcription conditions: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ and infinity. After completion of reverse transcription, 50. mu.L of RNase Free dH was added2The cDNA was diluted O and stored at-20 ℃ for later use in assays.
2) Taking 1 mu L of cDNA of each group of cells as a template, taking GAPDH as an internal reference, and detecting the relative expression quantity of c-myc by qPCR. Wherein the sequence of the primer for detecting the c-myc is as follows: 5'-CCTGGTGCTCCATGAGGAGA-3' (SEQ ID No.13) and 5'-TCCAGCAGAAGGTGATCCAGAC-3' (SEQ ID No. 14); the primer sequence of GAPDH is: 5'-TCTGACTTCAACAGCGACACC-3' (SEQ ID No.15) and 5'-CTGTTGCTGTAGCCAAATTCGT-3' (SEQ ID No. 16); setting reaction conditions: 30s at 95 ℃, 1 cycle, 30s at 55 ℃, 40 cycles at 95 ℃, 5s at 60 ℃, 1min at 95 ℃ and 15s at 95 ℃. The relative expression amounts of the c-myc genes of the hepatocyte L02 and the c-myc gene silencing cell strain are detected by using SYBR Primescript RT-PCR Kit, and the qPCR result is shown in FIG. 7. The ordinate of FIG. 7 shows the relative expression level of the c-myc gene.
As can be seen from FIG. 7, the c-myc gene expression of the hepatocyte L02 strain after c-myc gene silencing is obviously inhibited, and the gene expression level is reduced by 81% compared with that of hepatocyte L02.
(3) Western blot identification of c-myc gene silencing cell strain
Respectively taking 1 bottle (25 cm) of hepatocyte L02 and c-myc gene-silenced cell2Cell culture flask), removing culture medium, washing with cold PBS 3 times, adding 200 μ L cell lysate, scraping the lysed cells from the flask wall rapidly with cell scraper, collecting protein into EP tube of 500 μ L, continuously lysing at 4 deg.C for 30min, 12000rpm, centrifuging at 4 deg.C for 20min, finally adding 5 xSDS-PAGESAmple Loading Buffer, denaturing at 100 deg.C for 5min, and performing 12% SDS-polyacrylamide gel electrophoresis; electrically transferring the protein onto a PVDF membrane, and sealing with 5% skimmed milk powder for 1 h; respectively adding a c-myc antibody and a GAPDH antibody, incubating at 4 ℃ at room temperature overnight, washing the membrane for 3 times by TBST (tert-butyl-transferase) once every 10min, adding a secondary antibody, incubating at room temperature for 1h, washing the membrane for 3 times by TBST once every 10min, adding a Westernblot chemiluminescence reagent, and performing imaging analysis; the Western blot results are shown in FIGS. 8 to 9, wherein 1 in FIG. 9 indicates an untreated hepatocyte L02 group, 2 indicates a hepatocyte L02 group interfered by c-myc-shRNA2, and the ordinate of FIG. 9 indicates the relative content of c-myc. As can be seen from FIGS. 8 to 9, the Western blot result and the Q-PCR result correspond to that the c-myc protein expression of the L02 cell strain after c-myc gene silencing is reduced by 70%. The above results indicate that the c-myc gene-silenced cell strain was successfully constructed.
Example 4
Specific inhibition of the effects of c-myc Gene expression on apoptotic genes
The hepatocyte L02, the c-myc gene-silenced cell constructed in example 3, was used as the experimental subject, and the PM2.5 high dose (50. mu.g/mL) and the positive control Cr were used6+(10 mu M) is infected with the virus for 24h, and a blank control group is set up at the same time; fluorescent probeDetecting the relative expression quantity of the oncogenes (c-myc, c-fos, k-ras, p53) and the apoptosis genes (Caspase-3, Caspase-8 and Caspase-9) by quantitative PCR, wherein the primer sequence of the c-myc of the Q-PCR is shown as SEQ ID No.13 and SEQ ID No.14, and the primer sequence of the c-fos is as follows: 5'-TCTTACTACCACTCACCCGCAGAC-3' (SEQ ID No.17) and 5'-GGAATGAAGTTGGCACTGGAGA-3' (SEQ ID No.18), the primer sequences for k-ras are: 5'-GCGTAGGCAAGAGTGCCTTGA-3' (SEQ ID No.19) and 5'-GACCTGCTGTGTCGAGAATATCCA-3' (SEQ ID No.20), the primer sequence of p53 is: 5'-AGAGCTGAATGAGGCCTTGGAA-3' (SEQ ID No.21) and 5'-GAGTCAGGCCCTTCTGTCTTGAAC-3' (SEQ ID No.22), the primer sequence of Caspase-3 is: 5'-GACTCTGGAATATCCCTGGACAACA-3' (SEQ ID No.23) and 5'-AGGTTTGCTGCATCGACATCTG-3' (SEQ ID No.24), the primer sequence of Caspase-8 is: 5'-CAAATGCAAACTGGATGATGAC-3' (SEQ ID No.25) and 5'-AGCAGGCTCTTGTTGATTTGG-3' (SEQ ID No.26), the primer sequence of Caspase-9 is: 5'-GCCCATATGATCGAGGACATCCA-3' (SEQ ID No.27) and 5'-CACAACTTTGCTGCTTGCCTGTTAG-3' (SEQ ID No. 28). The results of the fluorescent quantitative PCR are shown in FIGS. 10 to 16.
FIG. 10 is a graph showing the results of the effect of PM2.5 on c-fos gene expression; FIG. 11 is a graph of the results of the effect of PM2.5 on c-myc gene expression; FIG. 12 is a graph showing the results of the effect of PM2.5 on k-ras gene expression; FIG. 13 is a graph showing the results of the effect of PM2.5 on the expression of p53 gene; FIG. 14 is a graph showing the results of the effect of PM2.5 on Caspase-3 gene expression; FIG. 15 is a graph showing the results of the effect of PM2.5 on Caspase-8 gene expression; FIG. 16 is a graph showing the results of the effect of PM2.5 on Caspase-9 gene expression; FIGS. 10 to 16 are graphs showing the relative expression amounts of c-fos, c-myc, k-ras, P53, Caspase-3, Caspase-8 and Caspase-9 in this order on the ordinate, and in FIGS. 10 to 16, ". X" indicates that P < 0.05 in comparison with the blank control group, ". X" indicates that P < 0.01 in comparison with the blank control group, ". #" indicates that P < 0.05 in comparison with the c-treated group, and, # indicates that P < 0.01 in comparison with the c-treated group.
As can be seen from FIGS. 10 to 16, the expression levels of the target genes in the c-myc gene-silenced cells were reduced by 257.7%, 62.0% and 93.2% for the oncogenes c-myc, c-fos and k-ras in the PM 2.5-infected group, respectively, as compared with that in the untreated normal hepatocyte L02; the expression of the cancer suppressor gene p53 is increased by 81.0 percent, and the apoptosis promoting gene CasThe expressions of Caspase-3, Caspase-8 and Caspase-9 are respectively reduced by 23.2%, 45.3% and 74.3%. Cr (chromium) component6+The expressions of the cancer genes c-fos, c-myc and k-ras of the infected positive control group are respectively reduced by 224.0%, 141.3% and 82.7%, the expression of the cancer suppressor gene p53 is increased by 68.3%, and the expressions of the apoptosis promoting genes Caspase-3, Caspase-8 and Caspase-9 are respectively reduced by 54.0%, 47.7% and 94.3%.
The above results show that: compared with an untreated normal hepatocyte L02, the hepatocyte L02 strain with c-myc gene silencing constructed in the example 3 has obvious changes of the expression of oncogenes and apoptosis genes after PM2.5 contamination treatment, which shows that the c-myc gene has an important effect on the apoptosis and canceration of PM 2.5-induced hepatocyte L02.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen disease prevention and control center (Shenzhen health inspection center, Shenzhen preventive medicine institute)
Construction method and application of shRNA (short hairpin ribonucleic acid) lentiviral expression vector for specifically inhibiting c-myc gene expression
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Claims (10)
1. An shRNA for specifically inhibiting c-myc gene expression is characterized by comprising a target sequence, a stem-loop structure, a complementary sequence of the target sequence and a termination site which are connected in sequence, wherein,
the target sequence is shown in SEQ ID NO.1, or,
the target sequence is shown in SEQ ID NO.2, or,
the target sequence is shown in SEQ ID No. 3.
2. An shRNA lentiviral expression vector for specifically inhibiting the expression of a c-myc gene, which comprises a vector and the shRNA according to claim 1 inserted in the vector.
3. An shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene according to claim 2, wherein the shRNA inserted into the vector is a double-stranded nucleotide fragment, and two strands of the double-stranded nucleotide fragment are respectively shown as SEQ ID No.4 to SEQ ID No. 5; or two chains of the double-stranded nucleotide fragment are respectively shown as SEQ ID No. 6-SEQ ID No. 7; or two chains of the double-stranded nucleotide fragment are respectively shown as SEQ ID No. 8-SEQ ID No. 9.
4. An shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene according to claim 2, wherein the vector is pLVX-shRNA1, the vector comprises a BamHI enzyme cutting site, an EcoRI enzyme cutting site and a KpnI enzyme cutting site, the shRNA comprises a viscous tail end of the BamHI enzyme cutting site and a viscous tail end of the EcoRI enzyme cutting site, and the shRNA is inserted between the BamHI enzyme cutting site and the EcoRI enzyme cutting site in the forward direction.
5. A method for constructing shRNA lentiviral expression vector for specifically inhibiting c-myc gene expression is characterized by comprising the following steps:
providing a double-stranded nucleotide fragment comprising the shRNA of claim 1 that specifically inhibits expression of the c-myc gene; and
and inserting the double-stranded nucleotide fragment into a lentiviral expression vector to obtain the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene.
6. The method for constructing shRNA lentiviral expression vector for specifically inhibiting the expression of c-myc gene according to claim 5, further comprising a step of transferring the lentiviral expression vector containing the double-stranded nucleotide fragment into a host cell after the step of inserting the double-stranded nucleotide fragment into the lentiviral expression vector.
7. The method for constructing the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene according to claim 6, wherein the host cell is a 293FT cell.
8. A recombinant engineering bacterium, which is characterized by containing at least one shRNA for specifically inhibiting the expression of the c-myc gene as claimed in claim 1, a shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene as claimed in any one of claims 2 to 4, and a shRNA lentiviral expression vector prepared by the method for constructing the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene as claimed in any one of claims 5 to 7.
9. The application of the shRNA specifically inhibiting the expression of the c-myc gene according to claim 1, the shRNA lentiviral expression vector specifically inhibiting the expression of the c-myc gene according to any one of claims 2 to 4, the shRNA lentiviral expression vector prepared by the construction method of the shRNA lentiviral expression vector specifically inhibiting the expression of the c-myc gene according to any one of claims 5 to 7 or the recombinant engineering bacterium according to claim 8 in the preparation of medicines for treating diseases related to abnormal expression of the c-myc gene.
10. A pharmaceutical composition, which comprises the shRNA for specifically inhibiting the expression of the c-myc gene according to claim 1, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene according to any one of claims 2 to 5, the shRNA lentiviral expression vector for specifically inhibiting the expression of the c-myc gene according to claim 7, or the recombinant engineering bacterium according to claim 8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111560374A (en) * | 2020-05-27 | 2020-08-21 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Construction and application of lentivirus and recombinant vector for specifically inhibiting c-fos gene expression |
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