CN113846099B

CN113846099B - siRNA for knocking down pig SAMHD1 gene expression, kit and application thereof

Info

Publication number: CN113846099B
Application number: CN202111117202.0A
Authority: CN
Inventors: 王�琦; 曹丽艳; 袁聪; 段月月; 索学鹏; 孔祥雨
Original assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Current assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-04-22
Anticipated expiration: 2041-09-23
Also published as: CN113846099A

Abstract

The invention belongs to the technical field of molecular biology and cell biology preparation, and discloses siRNA for knocking down pig SAMHD1 gene expression, a kit and application thereof, wherein a sense strand of the siRNA is a base sequence shown in SEQ ID NO.1, and an antisense strand of the siRNA is a base sequence shown in SEQ ID NO. 2. The siRNA for knocking down the pig SAMHD1 gene expression has high knocking efficiency, is suitable for pig-derived cell infection experiment operation taking lentivirus as a tool, can obviously improve the lentivirus infection efficiency of the pig-derived cell, and has wide application prospect.

Description

siRNA for knocking down pig SAMHD1 gene expression, kit and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and cell biology preparation, and relates to siRNA for knocking down pig SAMHD1 gene expression, a kit and application thereof.

Background

Lentiviruses belong to the family retroviridae, and are characterized by a long latency period which causes the individual to exhibit significant clinical symptoms more than the primary infection in macrophages and lymphocytes. The commonly used lentivirus vector is human immunodeficiency I type virus (HIV-1), and has the advantages of wide infection, effective infection of cells in a division stage and a stationary stage, insertion of large genetic fragments into host chromatin, long-term maintenance of stable transgene expression and the like, thereby being a favorable tool for introducing exogenous genes. The use of lentiviral vectors is limited by the infection efficiency of the lentivirus. In addition, no knockdown gene has been found that can increase the infection efficiency of different cells.

Disclosure of Invention

The invention aims to provide siRNA and a kit for knocking down pig SAMHD1 gene expression and application thereof, the gene knocking efficiency is high, and the lentivirus infection efficiency of different pig source cells can be obviously improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides siRNA for knocking down pig SAMHD1 gene expression, wherein a sense strand of the siRNA is a base sequence shown in SEQ ID NO.1, and an antisense strand of the siRNA is a base sequence shown in SEQ ID NO. 2.

The invention also provides a kit containing the siRNA.

Preferably, the kit further comprises a lentiviral packaging plasmid; the lentivirus packaging plasmid comprises three circular plasmids of pLenti-gfp, pMD2G and pSPAX 2.

Preferably, the lentiviruses are packaged using co-transfection with three circular plasmids in a molar ratio of 1:1:1, pLenti-gfp, pMD2G and pSPAX 2.

Preferably, the siRNA is transfected into cells requiring a final concentration of 150nM/μ L.

Preferably, the siRNA is transfected into the cell at the time of transfection within 12h before the cell is infected by the lentivirus.

The invention also provides application of the kit in improving the efficiency of infecting the porcine cells with the lentiviruses.

Compared with the prior art, the invention has the beneficial effects that:

the siRNA for knocking down the pig SAMHD1 gene expression has high knocking efficiency, is suitable for pig source cell infection experiment operation taking lentivirus as a tool, can obviously improve the lentivirus infection efficiency of different pig source cells (such as IPAM and BMDM), and has wide application prospect.

Drawings

FIG. 1 shows the efficiency of knocking down SAMHD1 gene expression in pigs transfected by different siRNAs.

FIG. 2 shows knockdown efficiency of siRNA-5 at different final concentrations of transfection.

FIG. 3 shows the knockdown efficiency of siRNA-5 after different times of transfection.

FIG. 4 shows the results observed by fluorescence inverted microscope 24h after cotransfection of plasmids.

FIG. 5 shows the fluorescent quantitative PCR amplification curve of siRNA-5.

FIG. 6 shows a melting curve of fluorescent quantitative PCR.

FIG. 7 shows the siRNA-5 knockdown efficiency assay results.

FIG. 8 shows the results of microscopic imaging of IPAM cells at the 36hpi time point.

FIG. 9 shows the results of microscopic imaging of IPAM cells at 48hpi time point.

FIG. 10 shows the results of microscopic imaging of IPAM cells at the 60hpi time point.

FIG. 11 shows the results of microscopic imaging of IPAM cells at the 72hpi time point.

FIG. 12 shows the results of microscopic imaging of IPAM cells at the 84hpi time point.

FIG. 13 shows the results of microscopic imaging of IPAM cells at the 96hpi time point.

FIG. 14 shows the results of microscopic imaging of IPAM cells at 108hpi time point.

Fig. 15 shows the results of flow cytometry cell clustering of IPAM cells in each well that were detoxified at MOI ═ 100.

Fig. 16 shows the results of flow cytometry cell clustering of BMDM cells in each well that were inoculated at MOI ═ 100.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. The reagents used in the following examples are all analytical grade reagents and are commercially available.

Example one pig SAMHD1 Gene knockdown efficiency assay

1.1 siRNA design

5 pairs of siRNA combinations, designated as siRNA-1, siRNA-2, siRNA-3, siRNA-4, and siRNA-5, were designed based on the published pig SAMHD1 gene sequence (NM-001292105.1) from the NCBI database and were synthesized by Shanghai Jima pharmaceutical technology, Inc., and the sequences are shown in Table 1.

TABLE 1siRNA designed according to pig SAMHD1 Gene

1.2 transfection with siRNA

When the IPAM cell density was 80%, siRNA was transfected into IPAM cells using the jetPRIME Polyplus Transfection kit (Ref #114-15/1.5mL), and the final concentrations of Transfection were selected as blank (NC), 50 nM/. mu.L, 100 nM/. mu.L, 150 nM/. mu.L and 200 nM/. mu.L, respectively. The medium was changed to an equal volume of serum-free RPMI 1640 medium 4-6h after siRNA transfection.

1.3 Lentiviral packaging

1.3.1 calculation of transfection volume of each plasmid

Three circular plasmid concentrations, pLenti-gfp, pMD2G and pSPAX2, were determined using a NanoDrop one ultramicro spectrophotometer. The mass of each plasmid to be transfected was calculated at a total mass of 10. mu.g transfected based on the nucleic acid chain length of each plasmid at a molar ratio of 1:1:1, and the results are shown in Table 2. A jetPRIME Polyplus Transfection kit containing siRNA and three circular plasmids was prepared.

TABLE 2 plasmid masses at transfection

1.3.2 Co-transfection of plasmids

A10 mL volume of 293T cell suspension at 2X 10⁵Placing the cells in a 10cm cell culture dish at a density of/mL until the cell number grows to 6X 10⁶Then, the jetPRIME Polyplus Transfection kit (Ref #114-15/1.5mL) was used to supply the kitThe plasmids were co-transfected into 293T cells according to the calculated volume, and the cell culture supernatant was replaced with 10mL serum-free DMEM medium 4h after transfection.

1.3.3 Lentiviral Collection and storage

And (3) after the plasmid is cotransfected for 48h, packaging the lentivirus, placing the packaged lentivirus in a refrigerator at the temperature of-80 ℃ by using a 10cm cell culture dish, repeatedly freezing and thawing, collecting a culture dish liquid phase in a sterile 15mL centrifuge tube, centrifuging at 2000rpm for 5min, and collecting a supernatant. Filtering the supernatant with 0.45 μm filter membrane, and packaging in sterile 1.5ml microfuge tube, or storing in-80 deg.C refrigerator.

1.4 lentivirus infection of IPAM cells

The porcine source cells to be treated in the experiment are respectively infected by the lentivirus 12h, 24h and 36h after siRNA transfection. Lentivirus-gfp was used as the positive control for the target experiment.

1.4.1siRNA transfection IPAM cell suspension was transfected at 2X 10 volumes of 2mL per well⁵The density of each/mL is respectively placed in a 6-well cell culture plate, 12mL is used in 6 wells, and when the cell density grows to about 80%, siRNA provided by a jet PRIME Polyplus Transfection kit (Ref #114-15/1.5mL) is used for transfecting siRNA provided by the kit into 3-well cells at a final concentration of 150 nM/. mu.L to serve as an experimental group; the cells in the other 3 wells were transfected with NC-siRNA at the same concentration as a control group.

The nucleic acid sequence of NC-siRNA is as follows: sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3', respectively; antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' are provided.

4h after siRNA transfection, the culture medium of each group of cells was replaced by an equal volume of serum-free RPMI 1640 medium.

1.5.2RNA extraction and reverse transcription

After 12h of transfection, cell pellets from each well were collected, and RNA from the cell pellets was extracted by TRIZOL lysis method and dissolved in 30. mu.L of RNase-free water.

Using reverse transcription kit (TAKARA PrimeScript)^TMRT Master Mix, RR036A) reverse transcribe the extracted RNA into cDNA, either for immediate use or for storage at-20 ℃.

1.5.3 fluorescent quantitation PCR detection of siRNA knockdown efficiency

TABLE 3 fluorescent quantitative PCR reaction System

And the detection primers comprise a SAMHD1 detection primer and a beta-actin detection primer.

The nucleic acid sequences of the SAMHD1 detection primer pair are as follows:

an upstream primer: 5'-TGGCAAATAAAAGAAATGGC-3'

An upstream primer: 5'-CTCACAGACACGGGCGAAT-3' are provided.

The beta-actin detection primer nucleic acid has the following sequence:

an upstream primer: 5'-CCGCACCACTGGCATTGTC-3'

A downstream primer: 5'-CTCCTTGATGTCCCGCACG-3' are provided.

1.5.4siRNA knockdown efficiency calculation

And (3) measuring the ct value of each sample according to the fluorescent quantitative PCR, calculating the siRNA knocking efficiency, and calculating the siRNA knocking efficiency by using GraphPad Prism 6 statistical analysis software.

1.5.5 results and analysis

The efficiency of knocking down the gene expression of the pig SAMHD1 by different siRNA transfections is shown in FIG. 1. As can be seen from FIG. 1, the knockdown efficiency of siRNA-5 was the best, and siRNA-5 was finally identified.

Knockdown efficiency of siRNA-5 at different final transfection concentrations is shown in FIG. 2. As can be seen from FIG. 2, siRNA transfection requires a final concentration of 150nM/μ L for knockdown efficiency and economy.

The knockdown efficiency of siRNA-5 after different times of transfection is shown in FIG. 3. As can be seen in FIG. 3, the percentage increase in GFP fluorescence was most efficient when the lentivirus was reinfected 12h after transfection, so the siRNA transfection was selected 12h before lentivirus infection.

The results of 24h after cotransfection of the plasmids observed by a fluorescence inverted microscope are shown in FIG. 4, wherein 1 is a control group and 2 is an experimental group, and cells are observed to emit green fluorescence under the fluorescence inverted microscope.

The fluorescent quantitative PCR amplification curve of siRNA-5 is shown in FIG. 5, wherein the red curve (upper part) in the figure represents that the detection primer targets beta-actin gene; the blue curve (below) represents its detection primer targeting SAMHD1 gene. As can be seen from FIG. 5, each sample to be tested exhibits an S-shaped amplification curve; negative samples had no amplification curve or ct values greater than 37.

The fluorescence quantitative PCR melting curve is shown in figure 6, wherein the red curve represents the detection primer targeting beta-actin gene; the blue curve represents its detection primer targeting SAMHD1 gene. As can be seen from FIG. 6, the two pairs of detection primers SAMHD1 and beta-actin each show independent peaks, and the peaks correspond to constant temperature.

The results of siRNA-5 knockdown efficiency assays are shown in FIG. 7. As can be seen from fig. 7, the knockdown efficiency of siRNA-5 reached 91.9%, the gene knockdown efficiency was high, and the difference was very significant compared to the control.

EXAMPLE two determination of lentivirus infection efficiency

2.1 Lentiviral packaging

See example 1.3.

2.2 transfection and grouping of siRNA IPAM cell suspension at 2X 10 at a volume of 1mL per well⁵The density of the/mL is set in 12-well cell culture plates, and 12mL is used in total for 1 plate. When the cell density is about 80%, transfecting siRNA provided by a jetPRIME Polyplus Transfection kit (Ref #114-15/1.5mL) into 6-well cells at a final concentration of 150 nM/. mu.L to serve as an experimental group; the cells in the other 6 wells were transfected with NC-siRNA at the same concentration as a control group. 4h after siRNA transfection, the culture medium was changed to serum-free RPMI 1640 medium for each group of cells.

2.3 lentivirus inoculation and grouping

To 3 of 6-well cells transfected with siRNA provided in the kit, Lenti-gfp was added at final MOI of 100, and this subset was designated as S1; in addition, the cells in the 3 holes are not subjected to virus inoculation treatment, and only DMEM serum-free culture medium with the same volume is added, wherein the grouping is marked as S0;

6-well cells transfected with NC-siRNA provided in the kit, to 3 of which was added Lenti-gfp at final MOI of 100, this grouping being designated NC 1; in addition, the 3-hole cells are not subjected to virus inoculation treatment, and only DMEM serum-free culture medium with the same volume is added, and the group is marked as NC 0;

after the lentivirus is inoculated for 2 hours, cell supernatants of all the wells are discarded, and the cells are replaced by a PRIM-1640 serum-free culture medium, wherein each well is 0.5 mL.

2.4 detection of lentivirus infection efficiency

Starting 36h after inoculation, the fluorescence change of the cells in each well is observed under a fluorescence inverted microscope every 12h, and images are retained.

Collecting cell precipitates of each well 108h after inoculation, resuspending the cell precipitates by PBS, centrifuging the cell precipitates to remove supernatant, repeating the steps for 3 times, then resuspending the cells of each well by using 4 percent paraformaldehyde with the volume of 300 mu L respectively, collecting the cell precipitates in a flow cytometry detection tube, placing the cell precipitates on ice in a dark place, and detecting FITC channel fluorescent signals by using a flow cytometer.

Experimental quantification data were analyzed and imaged by GraphPad Prism 6 statistical analysis software,

2.5 results and analysis

The results of microscopic imaging of IPAM cells at the 36hpi, 48hpi, 60hpi, 72hpi, 84hpi, 96hpi, and 108hpi time points for each experimental group are shown in FIGS. 8-14.

The flow cytometric grouping results of the individual well IPAM cells detoxified at Lentivirus-gfp at MOI of 100 are shown in FIG. 15. According to statistical analysis, the infection efficiency of IPAM cells reaches 40% at the MOI of 100 infection level, and the difference is significant compared with the control.

Example triple lentivirus infection of BMDM cells

Experimental procedures BMDM cells were infected according to example two. The results of flow cytometry cell clustering of BMDM cells in each well, which were inoculated at an MOI of 1000 with Lentivirus-gfp, are shown in FIG. 16. Statistical analysis showed that at MOI 100 infection level, the infection efficiency of BMDM cells also reached 25%, with a significant difference compared to the control.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences City agricultural research institute of China academy of agricultural sciences

<120> siRNA and kit for knocking down pig SAMHD1 gene expression and application thereof

<130> 2021

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> siRNA sense strand

<400> 1

gcaccugguu aauucuaaut t 21

<210> 2

<211> 21

<212> DNA

<213> siRNA antisense strand

<400> 2

auuagaauua accaggugct t 21

Claims

1. The application of the kit containing the siRNA for knocking down pig SAMHD1 gene expression in improving the efficiency of infecting pig cells with lentiviruses is characterized in that a sense strand of the siRNA is a base sequence shown in SEQ ID NO.1, and an antisense strand of the siRNA is a base sequence shown in SEQ ID NO. 2.

2. The use of claim 1, wherein the kit further comprises a lentiviral packaging plasmid; the lentivirus packaging plasmid comprises three circular plasmids of pLenti-gfp, pMD2G and pSPAX 2.

3. The use according to claim 1, wherein the lentivirus is packaged using co-transfection with three circular plasmids in a molar ratio of 1:1:1, pLenti-gfp, pMD2G and pSPAX 2.

4. The use of claim 1, wherein the siRNA is transfected into a cell at a final concentration of 150nM/μ L.

5. The use of claim 1, wherein the siRNA is transfected into the cell at a time prior to 12 hours prior to infection of the cell by the lentivirus.