CN113930451B - Report system for screening negative regulatory factors of interferon signal path and construction method thereof - Google Patents

Report system for screening negative regulatory factors of interferon signal path and construction method thereof Download PDF

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CN113930451B
CN113930451B CN202111167783.9A CN202111167783A CN113930451B CN 113930451 B CN113930451 B CN 113930451B CN 202111167783 A CN202111167783 A CN 202111167783A CN 113930451 B CN113930451 B CN 113930451B
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gene
interferon
negative regulatory
screening
sequence
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CN113930451A (en
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穆昕
杨晗
赵振翔
朱紫祥
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Tianjin University
Lanzhou Veterinary Research Institute of CAAS
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Lanzhou Veterinary Research Institute of CAAS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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    • C12N2510/00—Genetically modified cells

Abstract

The application belongs to the technical field of biology, and particularly relates to a reporting system for screening negative regulatory factors of an interferon signal path and a construction method thereof. The reporter system for screening negative regulatory factors of an interferon signal pathway comprises an interferon promoter coupled to a coding region of a resistance gene such that expression of the resistance gene is upregulated by activation of an intracellular interferon pathway. Under interferon treatment, the intracellular interferon pathway activation receptor will be up-regulated in expression, and thus more susceptible to loss of function of the negative regulatory factor. Most cells without the negative regulatory factor knocked out will not have an interferon pathway activated; cells in which the negative regulator encodes a gene knockout will activate the expression of the resistance gene due to activation of the interferon pathway. Screening and identification of negative regulatory factors in the interferon activation pathway are achieved in combination with the gene knockout library.

Description

Report system for screening negative regulatory factors of interferon signal path and construction method thereof
Technical Field
The application belongs to the technical field of biology, and particularly relates to a report system for screening negative regulatory factors of an interferon signal path and a construction method thereof.
Background
The interferon signal pathway plays an important role in resisting infection of pathogenic microorganisms, maintaining cell homeostasis, body health and the like, and is an important component of natural immunity. Abnormal activation of this pathway triggers the onset of inflammatory responses and autoimmune diseases in the body and is therefore critical for its proper regulation. Among these, negative regulatory factors play a key role: by modulating intracellular DNA, RNA metabolism, etc., silencing of this pathway is maintained in a non-infectious state.
Currently, screening and determination of negative regulatory factors are mainly divided into two cases: the first is obtained by high throughput screening based on differential expression of interferon pathway genes, which are then identified by functional experiments, e.g., lnc-Lsm3b 1 However, the efficiency of the discovery approach is low, the time consumption is long, the discovered differential expression genes do not necessarily exert negative regulation function, and the identification difficulty is increased. The second is that on the basis of gene discovery, the gene is identified as having negative regulation function of interferon pathway activation through the verification of subsequent researches, such as TREX1, RNASEH2, SAMHD1 and the like 2 However, this method has problems such as high cost, low efficiency, and long time. There is therefore a current lack of simple screening systems for efficient, high throughput of new negative regulatory factors.
In view of the important roles of research on negative regulatory factors in theoretical cognition and clinical application and the defects of the existing functional screening method, the application constructs a screening system for screening the negative regulatory factors of an interferon signal pathway, wherein the screening system is HeLa cells integrated with a reporting system of a resistance gene started by an interferon promoter, the whole genome knockout cell bank is constructed on the basis, the whole genome knockout cell bank is cultured in a culture medium containing interferon and antibiotics corresponding to the resistance gene, and if the gene knockout cell survives in the culture medium containing the antibiotics corresponding to the resistance gene, the gene knockout cell is the cell of which the gene is knocked out by the negative regulatory factor of the interferon signal pathway, namely, the knocked-out gene is judged to be the negative regulatory factor of the interferon signal pathway. The reporting system can solve the screening problem of negative regulatory factors in the interferon activation pathway.
Disclosure of Invention
In view of the above problems, the present application provides a report system for screening negative regulatory factors of an interferon signal pathway and a construction method thereof, which specifically includes the following steps:
in a first aspect, the present application provides a reporter vector for screening for negative regulators of the interferon signaling pathway, the reporter vector comprising: a gene combination element comprising an interferon promoter and a resistance gene, an expression vector, and/or a reporter gene element; the interferon promoter is positioned at the 5' -end of the resistance gene and is used for promoting the expression of the resistance gene; and the interferon promoter is opposite to the promoter for genome transcription of the expression vector.
Preferably, the gene combination element further comprises a Kozac sequence, and/or an SV40 poly a signal sequence; the 3 '-end of the Kozac sequence is connected with the 5' -end of the start codon of the resistance gene; the 5 'end of the SV40 poly A signal sequence was ligated to the 3' end of the resistance gene terminator.
Preferably, the resistance gene is an aminoglycoside antibiotic resistance gene.
Preferably, the antibiotic resistance genes include a neomycin resistance gene (NeoR), a hygromycin B resistance gene (hygr omycin resistant gene).
Preferably, the interferon promoter is a type I interferon pathway promoter.
Preferably, the interferon promoter is an IFN- β promoter that initiates transcription of the IFN- β gene.
Preferably, the IFN- β promoter has a gene sequence as set forth in SEQ ID NO. 2.
Preferably, the expression vector is an FG12 vector, and the gene combination element is inserted into the FG12 vector.
Preferably, the insertion mode of the gene combination element is as follows: after introduction of XhoI and XbaI sequences at the 5 'and 3' ends of the gene combination element containing the gene expressing the interferon promoter and the resistance gene, respectively, it was inserted into FG12 vector.
Preferably, the reporter is a luciferase reporter.
Preferably, the reporter gene comprises an eGFP gene.
Preferably, the gene sequence of the gene combination element is shown as SEQ ID NO.1 or SEQ ID NO. 3.
In a second aspect, the present application provides an interferon pathway activation reporter system for screening for negative regulators of the interferon signal pathway, said reporter system being a host cell transfected with any of the reporter vectors of the first aspect above.
Preferably, the host cell is a cell containing the CGAS-STING pathway.
Preferably, the host cell is a HeLa cell.
In a third aspect, the present application provides a screening system for screening negative regulatory factors of an interferon signaling pathway, the screening system comprising the reporter system of the second aspect above and a sg RNA targeted to knock out each gene in the whole gene of the reporter system; or the screening system is a library of gene knockout cells obtained after knocking out each gene in the reporter system according to claim 9, respectively.
Preferably, the sgRNA is selected from the group consisting of human_geckov2_library_a; the whole Genome knockout text human_geckov2_library_a covers 65384 sgrnas, derived from Zhang Feng 2018 published literature Genome-scale crispr-Cas9 knockout and transcriptional activation screening.
In a fourth aspect, the present application provides a method of screening for negative regulatory factors of an interferon signaling pathway, the method comprising the steps of:
(1) Constructing a library of gene knockout cells according to the third aspect;
(2) Taking out the gene knockout cells in the gene knockout cell library respectively, adding interferon to induce for 24 hours, and culturing in a culture medium containing antibiotics corresponding to the resistance genes;
(3) And (3) result judgment: if the gene knockout cell exists in a culture medium containing antibiotics corresponding to the resistance genes and the interferon, the gene knockout cell is a cell in which the negative regulatory factor of the interferon signal path codes for gene knockout, and the knocked-out gene is judged to be the negative regulatory factor of the interferon signal path.
The beneficial effects of the application are as follows: the application provides a report system for screening negative regulation factors of an interferon signal path and a construction method thereof, wherein the report system can be used for screening and functional identification of the negative regulation factors of the interferon signal path, and is beneficial to large-scale screening of genes or medicines with negative regulation functions for activating the interferon path.
Drawings
FIG. 1 schematic diagram of cleavage sites on FG12 vector;
FIG. 2 is a view of the fluorescent microscope;
FIG. 3 IFNseta and ISG15 content detection results;
FIG. 4NeoR reports system expressed test results;
fig. 5 reports the results of the system verification experiment.
Detailed Description
The application is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the application easy to understand. The scope of the application is not limited to the examples described below.
Experimental cells, viral sources as described in the examples below:
other reagents in the experiment are common commercial reagents unless specifically stated otherwise; the procedure in the experiment is known in the art unless specifically indicated.
The present application will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present application, but are not intended to limit the application in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present application.
EXAMPLE 1 construction of reporter System for screening negative regulators of the interferon Signal pathway
Construction of IFN- β promoters and coding sequences
According to NCBI database, the sequence information of IFN- β DNA, including promoter sequence and downstream partial sequence, was queried. The sequence is displayed in the direction of 5'-3', and is a promoter sequence, a sequence between the promoter and a coding region, and a coding region sequence, wherein the specific gene sequence is shown as SEQ ID NO. 2.
2. Construction of Gene combinatorial elements for screening of negative regulatory factors of the Interferon Signal pathway
Synthesizing a gene combination element for screening negative regulatory factors of an interferon signal pathway, wherein the gene combination element sequentially comprises: an IFN- β promoter sequence, an entire sequence downstream of the IFN- β promoter up to the start codon, a K ozac sequence (GCCACC), a coding sequence for NeoR, a TGA terminator, an SV40 poly (A) signal sequence; the gene sequence of the gene combination element is shown as SEQ ID NO. 1.
And respectively introducing enzyme cutting sites XbaI and XhoI at the 5 'and 3' ends of the gene combination element to form double enzyme cutting gene fragments, wherein the double enzyme cutting gene fragments sequentially comprise: the cleavage site protecting base sequence aatt, xhoI recognition sequence, IFN- β promoter sequence, all sequences downstream of IFN- β promoter to the start codon, kozac sequence (G CCACC), neoR coding sequence, TGA terminator, SV40 poly (A) signal sequence, xbaI recognition sequence, cleavage site protecting base sequence ttaa. The gene sequence of the double enzyme cutting gene fragment is shown as SEQ ID NO. 3.
3. Construction of reporting vector
The double-restriction enzyme fragments prepared in the step 2 are inserted into XbaI and XhoI restriction enzyme sites (shown in figure 1) on the FG12 vector, so that the double-restriction enzyme fragments are reversely inserted into the FG12 vector, and a report vector for screening negative regulatory factors of an interferon signal path is constructed.
4. Construction and screening of reporting systems
Packaging the report vector prepared in the step 3 in HEK293T cells; collecting the supernatant containing viruses, infecting HeLa cells, carrying out cell dilution culture, and picking out a monoclonal containing eGFP signals, which is named HeLa-IFNb-NeoR; the monoclonal HeLa-IFNb-NeoR was subjected to expansion culture, and the cell line was confirmed to carry the eGFP tag on the FG12 vector by flow cytometry and fluorescent microscopy.
The result of eGFP expression analysis by HeLa-IFNb-NeoR is shown in FIG. 2, A is that cells are subjected to pancreatin digestion for flow cytometry; b is eGFP expression analysis of the cells circled in A, with the vertical axis representing the number of cells and the horizontal axis representing the eG FP intensity. C is the fluorescent microscope observation of the cell line, and the GFP signal is detected. The results indicate that the reporting system carries the eGFP tag on FG12 vector.
Results validation of IFN- β expression levels
The HeLa-IFNb-NeoR was subjected to an interferon pathway activation assay to detect the response of the cell to interferon pathway activation: transfecting plasmid and Empty Vector (EV) for expressing MAVS, extracting total RNA after 24 hours for RT-qPCR, and analyzing cell RNA by using RT-qPCR; wherein following transfection of the MAVS plasmid, the cell expresses a MAVS protein, the overexpression of which is capable of aggregating into a complex on mitochondria, activating the function of a downstream interferon activator, forcing the cell to activate the interferon signaling pathway.
The results are shown in FIG. 3, wherein A is the result of detection of IFN- β expression levels. The results indicate that in the case of IFN- β (shown as IF Nb in the figure) activation, both IFN- β and ISG15 in HeLa-IFNb-NeoR are activated to a relatively weak extent as compared to normal HeLa, suggesting that the reporter vector competes with the endogenous IFNb gene for interferon-activating promoter.
NeoR reporter expression detection
After confirming that the cell line is able to activate the interferon pathway, a test of expression of the NeoR reporter system is performed: separately transfecting HeLa and HeLa-IFNb-NeoR cells with MAVS expression plasmids, taking no treatment as a control, and carrying out dilution culture on the sterilized cells after 48 hours, and adding 2000 mug/ml G-418 (neomycin) into a culture medium; the surviving cells were then subjected to crystal violet staining.
The results are shown in FIG. 4, wherein A is the G-418 treatment of HeLa and HeLa-IFNb-NeoR cells; b is that H eLa and HeLa-IFNb-NeoR cells are transfected with plasmids expressing MAVS and then G-418 treatment is carried out. The results showed that both normal HeLa and HeLa-IFNb-NeoR cells were all killed in the control group (as shown in fig. 4 a); after transfection of the M AVS expression plasmid, the intracellular interferon pathway is activated, and the expression of NeoR is also activated in HeLa-IFNb-NeoR cells, so that the cells can still survive in the presence of G-418 with the same concentration; whereas normal HeLa cells were unable to survive the G-418 treated culture conditions following MAV S expression (as shown in FIG. 4B).
By experimental comparison, heLa-IFNb-NeoR has an activating reaction to an interferon pathway, can be induced by interferon to initiate expression of NeoR resistance protein, and can survive in a medium containing G-418. The screening is completed by the report system construction.
Example 2 verification test
To further confirm that the response of the reporter cell line to interferon pathway activation can be used for screening of negative regulatory factors, knockout of ADAR1 genes was performed by CRISPR-Cas9 means. ADAR1 is a deaminase that chemically modifies double-stranded RNA, and is capable of modifying adenylate to hypoxanthine, thereby disrupting a present in the double-stranded RNA: u pairing inhibits recognition of double stranded R NA by the cell. ADAR1 can inhibit endogenous double-stranded RNA from being incorrectly recognized as virus double-stranded RNA, so that the internal environment steady state of cells is effectively protected, and the abnormal activation of an interferon channel is inhibited. The knockout of ADAR1 can activate a new round of interferon signal channels of cells under the condition of interferon stimulation, which indicates that an interferon recognition system of the cells performs error recognition on endogenous unmodified double-stranded RNA after up-regulated expression.
By constructing sgRNA for stably expressing and targeting ADAR1 genes, a knockout cell line is constructed on the basis of HeLa-IFNb-NeoR. The reduction in RNA levels of ADAR1 was confirmed by RT-qPCR. The expression level of the intracellular interferon activation system is improved by interferon treatment, and then the surviving cell clone is obtained by G-418 drug screening. The surviving cells are those cells in which the interferon pathway is activated resulting in expression of NeoR (neomycin resistance gene).
The results are shown in FIG. 5: wherein A is total RNA extraction of HeLa-IFNb-NeoR and HeLa-IFNb-NeoR-ADAR1KO cells, and then RT-qPCR is carried out to detect the ADAR1 expression level; b is that HeLa-IFNb-NeoR and HeLa-IFNb-NeoR-ADAR1KO cells are subjected to interferon treatment and then G-418 treatment. The results indicate that in ADAR1-KO treated cells, surviving cell clones can be seen.
The results show that the screening system for the negative regulatory factor activated by the interferon pathway can positively realize the screening of the negative regulatory factor activated by the interferon pathway, and has the advantages of rapidness, accuracy and the like.
Sequence listing
<110> university of Tianjin
Lanzhou Institute of veterinary medicine, Chinese Academy of Agricultural Sciences
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Claims (1)

1. A method of screening for negative regulatory factors of an interferon signaling pathway, comprising the steps of:
(1) Constructing a gene knockout cell bank;
(2) Taking out the gene knockout cells in the gene knockout cell library respectively, adding interferon to induce for 24 hours, and culturing in a culture medium containing antibiotics corresponding to the resistance genes;
(3) And (3) result judgment: if the gene knockout cell survives in a culture medium containing antibiotics corresponding to the resistance gene and the interferon, the gene knockout cell is a cell in which the negative regulatory factor of the interferon signal path codes for gene knockout, and the knocked-out gene is judged to be the negative regulatory factor of the IFN-beta interferon signal path;
the gene knockout cell bank is as follows: a gene knockout cell bank obtained after knocking out each gene in the report system; the report system is HeLa cells transfected with a report vector for screening negative regulatory factors of an interferon signal path; the report carrier comprises: a gene combination element comprising an IFN- β promoter and a resistance gene, and an FG12 expression vector; the gene combination element is inserted into an FG12 expression vector; the gene combination element comprises the following components in sequence according to the 5'-3' direction: an IFN- β promoter sequence, all sequences downstream of the IFN- β promoter up to the start codon, the coding sequence for the Kozac sequence GCCACC, neoR, the TGA terminator, the SV40 poly (A) signal sequence; the gene sequence of the gene combination element is shown as SEQ ID NO.1 or SEQ ID NO. 3.
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