CN116144707A - Application of reagent for over-expressing HS3ST5 gene in promotion of virus infection and recombinant CHO-K1 cell line - Google Patents
Application of reagent for over-expressing HS3ST5 gene in promotion of virus infection and recombinant CHO-K1 cell line Download PDFInfo
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Abstract
The invention provides an application of a reagent for over-expressing HS3ST5 genes in promoting virus infection and a recombinant CHO-K1 cell line constructed by the reagent, and belongs to the technical field of genetic engineering. The invention provides a recombinant CHO-K1 cell line expressing HS3ST5, which is obtained by cloning HS3ST5 fragments into a lentiviral vector to construct a recombinant lentiviral plasmid, and then infecting CHO-K1 cells by virus rescue packaging lentivirus. Recombinant CHO-K1 cells stably expressing HS3ST5 can promote adsorption, internalization, gene replication and progeny virus generation of FMDV, which provides tools for researching virus pathogenic mechanisms and provides a basis for subsequent preparation of virus infection cell models.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of a reagent for over-expressing HS3ST5 genes in promoting virus infection and a recombinant CHO-K1 cell line.
Background
Heparan Sulfate (HS) is a class of linear sulfated heterogeneous polysaccharides that are found in large quantities on mammalian cell surfaces and in the extracellular matrix. HS is capable of binding to a variety of ligands and is considered to be a non-specific cell surface receptor. HS has been found to assist in viral infection by unique monosaccharide sequences, and thus, HS can act as a specific receptor for target cells infected by viruses. Further studies have shown that HS is involved in the viral adsorption and entry process, facilitating viral proliferation within host cells. Herpes simplex virus type 1 (HSV-1) and Foot and Mouth Disease Virus (FMDV) can infect target cells using HS as a viral receptor. Various viruses, such as dengue virus (DENV) and Human Immunodeficiency Virus (HIV), utilize specific HS polysaccharide structures to infect cells. In addition, HS has also been demonstrated to bind directly to angiotensin converting enzyme 2 (ACE 2) as an accessory receptor for the currently prevailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), promoting the adsorption and invasion of SARS-CoV-2 into host cells. HS biosynthesis modification enzymes affect the binding specificity and biological function of HS proteins by modifying the degree of HS sulfation, polysaccharide structure, and the like. Heparan sulfate 3-O-sulfate transferase (HS 3 ST) is a key enzyme in HS biosynthesis that catalyzes the transfer of sulfate groups from a sulfate donor to the 3-OH position of glucosamine to form 3-O-sulfated HS. HS3ST includes 7 isomers, of which HS3ST5 is the most biologically active. At present, the research on the influence of HS3ST5 on virus infection is few, and no report exists on the construction and application of a cell line for stably expressing HS3ST5.
Picornaviruses play an important role in medicine and veterinary medicine, and their research results have important reference values. FMDV is a classical picornavirus, often used as a model virus to study virus-host interactions. The utilization of host cell surface receptors by viruses not only determines the invasion efficiency and replication capacity of the viruses, but also determines the host range and pathogenicity of the viruses. During the adaptation of FMDV field strains to cell culture, the virus gains the ability to infect cells using HS as a receptor. FMDV recognizes and binds to HS receptors on the surface of cell membranes and adsorbs onto host cells, followed by HS-mediated endocytosis into the cells. FMDV and HS interaction studies focus on the efficiency and capacity of HS utilization of key amino acid residues on different FMD strains and on the same strain. The effect of the HS modifying enzyme HS3ST5 on FMDV replication is not yet clear.
Disclosure of Invention
Accordingly, the present invention is directed to the use of an agent that overexpresses the HS3ST5 gene to promote viral adsorption and invasion of host cells.
The invention provides application of a reagent for over-expressing HS3ST5 genes in promoting virus infection.
Preferably, the nucleotide sequence of the HS3ST5 gene is shown as SEQ ID NO. 1.
Preferably, the agent that overexpresses the HS3ST5 gene comprises a recombinant lentiviral plasmid that overexpresses the HS3ST5 gene.
Preferably, the increasing viral infectivity includes increasing the ability of the virus to adsorb and invade host cells, increasing viral gene replication and progeny virus replication levels.
Preferably, the virus comprises a virus that invades cells using HS receptors.
Preferably, the virus that invades cells using HS receptor includes one or more of foot-and-mouth disease virus, severe acute respiratory syndrome coronavirus and herpes simplex virus.
The invention provides a recombinant cell line for stably expressing HS3ST5, which is used for over-expressing HS3ST5 protein.
The invention provides a construction method of the recombinant cell line for stably expressing HS3ST5, which comprises the following steps:
1) Constructing a recombinant lentiviral plasmid over-expressing HS3ST5 gene;
2) Co-transfecting the recombinant lentiviral plasmid and the auxiliary plasmid which are used for over-expressing the HS3ST5 gene in the step 1) into a co-transfected cell to rescue to obtain the recombinant lentivirus which is used for over-expressing the HS3ST5 gene;
3) The recombinant lentivirus obtained by rescue is infected with CHO-K1 cells, and a recombinant cell line which stably expresses HS3ST5 is obtained by screening.
Preferably, in the construction method in the step 1), the HS3ST5 gene is amplified by using a primer containing Xba I and NotI cleavage sites, and the obtained amplified fragment and lentiviral vector pLOV-CMV-EGFP are respectively subjected to double cleavage by Xba I and NotI, the cleavage fragment is connected with a linearization vector, and the recombinant lentiviral plasmid over-expressing the HS3ST5 gene is obtained by identification.
The invention provides an application of the recombinant CHO-K1 cell line stably expressing HS3ST5 or the recombinant CHO-K1 cell line stably expressing HS3ST5 obtained by the construction method in preparing virus infection cell models.
The invention provides application of a reagent for over-expressing HS3ST5 genes in promoting virus infection. Experiments prove that the HS3ST5 fragment is obtained through amplification by RT-PCR technology, the recombinant lentiviral plasmid is constructed by cloning the fragment into a lentiviral vector, then the packaged lentivirus is saved by the virus, and then the CHO-K1 cell is infected, so that the recombinant CHO-K1 cell line expressing the HS3ST5 is obtained. The FDMV is taken as a virus model, the adsorption, internalization, gene replication and progeny virus levels of FMDV on recombinant CHO-K1 cells are measured, and finally the influence of the recombinant cells on FMDV replication capacity is evaluated, so that the result shows that the recombinant CHO-K1 cells stably expressing HS3ST5 can promote the adsorption, internalization, gene replication and progeny virus generation of FMDV. It can be seen that overexpression of the HS3ST5 gene can influence adsorption of FMDV to cells, and promote infection of FMDV. The method provides a tool for researching the pathogenic mechanism of the virus and provides a basis for preparing a virus infection cell model subsequently.
Drawings
FIG. 1 shows the results of HS3ST5 gene amplification;
FIG. 2 shows the expression results of recombinant lentiviral plasmids with EGFP tags in HEK-293T cells;
FIG. 3 shows the expression results of EGFP in a monoclonal recombinant CHO-K1 cell line;
FIG. 4 shows the results of HS3ST5 protein expression analysis in CHO-HS3ST5-OE-1 recombinant cell lines;
FIG. 5 shows the detection of HS3ST5 mRNA level in CHO-HS3ST5-OE-1 recombinant cell lines;
FIG. 6 shows the results of growth curves of recombinant CHO-K1 cells stably expressing HS3ST 5;
FIG. 7 shows the specific growth rate results of recombinant CHO-K1 cells stably expressing HS3ST 5;
FIG. 8 shows the results of HS3ST5 protein expression during passage of recombinant CHO-K1 cells;
FIG. 9 is a graph showing the effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV adsorption;
FIG. 10 is the effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV internalization;
FIG. 11 is the effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV gene replication;
FIG. 12 shows the effect of recombinant CHO-K1 cells stably expressing HS3ST5 on proliferation of sub-fmDV.
Detailed Description
The invention provides application of a reagent for over-expressing HS3ST5 genes in promoting virus infection.
In the present invention, the nucleotide sequence of the HS3ST5 gene is preferably shown as SEQ ID NO. 1. The agent that overexpresses the HS3ST5 gene preferably comprises a recombinant lentiviral plasmid that overexpresses the HS3ST5 gene. The virus preferably comprises a virus that invades cells using HS receptors. In the examples of the present invention, foot-and-mouth disease virus was used as a representative of viruses that invade cells using HS receptors, and the effect of agents that overexpress HS3ST5 gene on the ability of the viruses to infect was studied. In the embodiment of the invention, the result shows that the over-expression of the HS3ST5 gene is beneficial to promoting the virus adsorption and invasion of host cells and improving the virus gene replication and progeny virus replication level.
The invention provides a recombinant CHO-K1 cell line for stably expressing HS3ST5, wherein the recombinant CHO-K1 cell line overexpresses HS3ST5 protein. The amino acid sequence of the HS3ST5 protein is shown as SEQ ID NO: 2.
The invention provides a construction method of a recombinant CHO-K1 cell line for stably expressing HS3ST5, which comprises the following steps:
1) Constructing a recombinant lentiviral plasmid over-expressing HS3ST5 gene;
2) Co-transfecting the recombinant lentiviral plasmid and the auxiliary plasmid which are used for over-expressing the HS3ST5 gene in the step 1) into a co-transfected cell to rescue to obtain the recombinant lentivirus which is used for over-expressing the HS3ST5 gene;
3) The recombinant lentivirus obtained by rescue is infected with CHO-K1 cells, and a recombinant CHO-K1 cell line which stably expresses HS3ST5 is obtained by screening.
In the construction method, the HS3ST5 gene is preferably amplified by using a primer containing Xba I and Not I cleavage sites, the obtained amplified fragment and a lentiviral vector pLOV-CMV-EGFP are respectively subjected to double cleavage by the Xba I and Not I, the cleavage fragment is connected with a linearization vector, and the recombinant lentiviral plasmid over-expressing the HS3ST5 gene is obtained through identification. The primers containing Xba I and NotI cleavage sites comprise a forward primer with a nucleotide sequence shown as SEQ ID NO. 3 (CGTCTAGAATGAAAAAGCGTAGTAGTG) and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 (ATATGCGGCCGCGGGCCAGTTCAATGT). The conditions of the amplification are preferably 94℃for 5min of pre-denaturation; denaturation at 94℃for 1min, annealing at 57℃for 1min, and extension at 72℃for 2.5min. The method of the present invention is not particularly limited, and the method of cleavage and ligation may be any known method known in the art.
In the present invention, the co-transfected cells preferably comprise components in parts by mass: 10 parts of recombinant lentiviral plasmid, 7.5 parts of psPAX2 helper plasmid, 2.5 parts of pMD2.G helper plasmid. The transfection reagent is preferably Lipofectamine 2000 when co-transfecting cells. The invention is not limited to the types of cells, and cells known in the art can be used, and HEK-293T cells are used as infected cells in the embodiment of the invention.
In the present invention, rescue of the over-expressed HS3ST5 recombinant lentivirus is preferably selected with 2. Mu.g/mL puromycin. Through RT-qPCR detection, the level of HS3ST5 mRNA in the obtained screened cell line is obviously increased, which indicates that the recombinant cell line which over-expresses HS3ST5 is successfully constructed.
In the present invention, the level of the HS3ST5 protein expressed by the recombinant CHO-K1 cell line of the 5 th and 20 th generation of recombinant cell lines is not significantly different by Western Blot analysis, which indicates that the recombinant CHO-K1 cell line can stably express the HS3ST5 protein. The results of the growth curve and the specific growth speed show that the stable expression of HS3ST5 does not substantially affect the growth performance of CHO-K1 cells.
In the present invention, recombinant CHO-K1 cells stably expressing HS3ST5 promote adsorption, internalization, gene replication and progeny virus production of FMDV.
The invention provides an application of the recombinant CHO-K1 cell line stably expressing HS3ST5 or the recombinant CHO-K1 cell line stably expressing HS3ST5 obtained by the construction method in preparing virus infection cell models.
The use of an agent for over-expressing the HS3ST5 gene according to the present invention for promoting viral adsorption and invasion of host cells is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction method of recombinant lentiviral plasmid over-expressing HS3ST5 gene
1. Material
1.1 cells, plasmids and viruses
BHK-21 cells were derived from China center for type culture collection, CHO-K1 cells were derived from U.S. Collection of standard organisms, HEK-293T cells were derived from China center for type culture collection, JM109 competent cells were purchased from TaKaRa, pMD2.G, psPAX2 and pLOV-CMV-EGFP plasmids were purchased from Invitrogen, and classical O-type foot-and-mouth disease vaccine strain O/HN/CHA/93 (Cathay) was supplied by China national foot-and-mouth disease reference laboratory.
1.2 major reagents and instruments
Rabbit HS3ST5 polyclonal antibody was purchased from Novus corporation, murine β -actin monoclonal antibody was purchased from well-known century corporation, RNeasy Mini Kit was purchased from Qiagen corporation, phosphate Buffered Saline (PBS) was purchased from BI biosystems, tragacanth was purchased from MP Biomedicals corporation, high sugar DMEM, MEM medium, F-12K, lipofectamine 2000, opti-MEM medium and pancreatin was purchased from Invitro gen corporation, fetal Bovine Serum (FBS) and puromycin was purchased from Gibco corporation, plasmid miniprep Kit and plasmid miniprep Kit were purchased from the root biochemical technology (north) corporation, lentivirus rapid assay card was purchased from beijing bolong immunotechnology corporation, chamQ SYBR qPCR Master Mix was purchased from nuzano corporation.
1.3 primer design and Synthesis
Primer pairs were designed based on the HS3ST5 gene sequence on NCBI to perform RT-PCR amplification of HS3ST5 gene in CHO-K1 cells, with the designed upstream primer being 5'-CGTCTAGAATGAAAAAGCGTAGTAGTG-3' (SEQ ID NO: 3) and downstream primer being 5'-ATATGCGGCCGCGGGCCAGTTCAATGT-3' (SEQ ID NO: 4). Primers for GAPDH reference gene: the upstream primer was 5'-CAAGAAGGTGGTGAAGCA-3' (SEQ ID NO: 5) and the downstream primer was 5'-AAGTGGAAGAGTGAGTGTC-3' (SEQ ID NO: 6) all of which were synthesized by Jin Weizhi Biotechnology Inc.
2. Method of
2.1 Amplification of HS3ST5 Gene
The primers designed and synthesized as described above were used to amplify the HS3ST5 gene. The PCR reaction system is shown in Table 1, and the reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 57℃for 1min, extension at 72℃for 2.5min, and cycling for 35 times; and extending at 72 ℃ for 10min. The PCR product was subjected to 1% agarose gel electrophoresis and then subjected to DNA agarose gel recovery, and the recovered product was sent to Jin Weizhi Biotechnology company for sequencing.
TABLE 1 RT-PCR System
2.2 construction of recombinant lentiviral plasmids overexpressing HS3ST5
The lentiviral vector pLOV-CMV-EGFP was digested with Xba I and NotI to recover 8000bp fragment. The recovered fragment and the HS3ST5 amplified fragment were ligated to construct a recombinant lentiviral plasmid, designated pLOV-EGFP-CHO-HS3ST5. The pLOV-EGFP-CHO-HS3ST5 plasmid is subjected to preliminary enzyme digestion identification, positive plasmids are sent to Jin Weizhi biotechnology company for sequencing, and sequencing primers are as follows: 5'-CGGTGAATGCTGGTGGCATC-3' (SEQ ID NO: 5). The correct positive recombinant plasmids and helper plasmids pMD2.G and psPAX2 were identified by mass extraction.
3. Results
Amplification of 3.1HS3ST5 Gene
The HS3ST5 gene was amplified using primers with RNA in CHO-K1 cells as template. Agarose gel electrophoresis results showed successful amplification of the HS3ST5 gene in CHO-K1 cells. The electrophoresis chart is shown in FIG. 1, and the size of the amplified product band is about 1000 bp.
3.2 construction and identification of recombinant lentiviral plasmids
The pLOV-CMV-EGFP vector was digested with Xba I and Not I, 2 fragments were observed by agarose gel electrophoresis, 8000bp fragments were recovered, and ligated with the amplified fragment of HS3ST5. After preliminary identification of the ligation products by agarose gel electrophoresis, the positive clones were sent for identification, and the results indicate that the recombinant lentiviral plasmid pLOV-EGFP-CHO-HS3ST5 was successfully constructed. Positive plasmids with correct sequencing are greatly extracted and stored for standby.
Example 2
Construction method of recombinant CHO-K1 cell line
1. Rescue of overexpressed HS3ST5 recombinant lentiviruses
Normal HEK-293T cells were plated in 10cm cell culture dishes and when the cells were well conditioned and at a density of 70%, plasmid transfection was performed using Lipofectamine 2000 transfection reagent (10 μg recombinant lentiviral plasmid +7.5 μg psPAX2 helper plasmid +2.5 μg pmd2.G helper plasmid). After 6 hours, 4ml of high sugar DMEM complete medium was gently added to the cell culture dish, and the culture was carried out in a 37 ℃ incubator, during which the green fluorescence intensity of the transfected cells was observed multiple times using a fluorescence microscope, and after 48 hours, the cell supernatant was collected and filtered with a 0.45um filter. And (3) using a lentivirus rapid detection card to measure the titer of the lentivirus in the supernatant, and placing the filtered lentivirus liquid at the temperature of minus 30 ℃ for later use.
2. Determination of puromycin screening concentration
CHO-K1 cells were plated into six well plates and after cell densities were 90%, puromycin-treated cells at 1, 2, 3, 4, 5 and 6 μg/mL were added, respectively, and the treatment was re-dosed every 24 h. After 7d, observing the cell survival condition of the six-hole plate, wherein the lowest drug concentration without cell survival is the optimal concentration for puromycin screening.
3. Construction of recombinant CHO-K1 cell line stably expressing HS3ST5
Lentivirus and complete cell culture medium were mixed according to 1:1 volume of the mixture was mixed to prepare a mixed medium, and normal CHO-K1 was plated in a six-well plate and cultured with the mixed medium. After the cells grow into six pore plates, transferring the cells into a cell culture bottle, continuously adding the mixed culture medium for culture, and replacing the mixed culture medium every 24 hours. After 7d, the green fluorescence of the cells after lentiviral infection was observed by fluorescence microscopy. Cells after lentiviral infection were treated with the optimal concentration of puromycin and the complete medium with puromycin was changed every 24 h. CHO-K1 cells that survived 7d were almost all recombinant cells harboring the recombinant lentiviral plasmid. Recombinant CHO-K1 cells over expressing HS3ST5 were counted and diluted into single cells, which were then added to 96-well plates, and after growing into a pellet, the cells were observed microscopically for single clones. After the cells are full, the monoclonal recombinant cells are subjected to expansion culture and cryopreservation, and the expression level of HS3ST5 in the monoclonal recombinant cells is detected by Western Blot and real-time fluorescent quantitative PCR (RT-qPCR).
Results
Establishment and identification of recombinant CHO-K1 cell line stably expressing HS3ST5
HEK-293T cells were transfected with the positive recombinant lentiviral plasmid pLOV-EGFP-CHO-HS3ST5, and after 48 hours, the cells were observed with a fluorescence microscope for green fluorescence. As shown in FIG. 1, stronger green fluorescence was observed in transfected cells under a fluorescence microscope. Because the recombinant lentiviral plasmid carries the EGFP protein tag, the successful expression of the EGFP protein in the HEK-293T cell indicates that the recombinant lentiviral plasmid is successfully transfected into the HEK-293T cell, and the over-expression recombinant lentiviral rescue is successful. CHO-K1 cells were treated with graded concentration of puromycin and it was observed that all cells died after screening for puromycin at a minimum concentration of 2. Mu.g/mL for one week. Thus, the optimal puromycin concentration for screening CHO-K1 recombinant cell lines was determined to be 2. Mu.g/mL. After CHO-K1 cells were successfully rescued for recombinant lentiviral infection and screened for optimal concentration of puromycin, fluorescence microscopy was used to see if the cells were green fluorescent. As shown in FIG. 2, fluorescence microscopy observed that the recombinant lentivirus-infected CHO-K1 cells emitted stronger green fluorescence, indicating successful infection of the recombinant lentivirus by the CHO-K1 cells, indicating successful initial construction of the recombinant CHO-K1 cell line over-expressing HS3ST5. The constructed recombinant cell line was named CHO-HS3ST5-OE, and 3 monoclonal cell lines with stronger fluorescence were selected from the recombinant cell line for cryopreservation (see FIG. 3). CHO-HS3ST5-OE-1 was selected for validation of HS3ST5 protein and mRNA expression levels. As shown in FIG. 4, the Western Blot results demonstrate that recombinant cell line CHO-HS3ST5-OE-1 successfully expressed the HS3ST5 protein compared to normal CHO-K1 cells. As shown in FIG. 5, the RT-qPCR results showed a significant increase in HS3ST5 mRNA levels in CHO-HS3ST5-OE-1 compared to normal CHO-K1 cells. The above results indicate that the establishment of the recombinant CHO-K1 cell line overexpressing HS3ST5 was successful.
Example 3
Growth curve and specific growth rate analysis of recombinant CHO-K1 cells stably expressing HS3ST5
After the recombinant CHO-K1 cells and normal CHO-K1 cells are fully grown, digesting the cells with pancreatin, counting the cell suspension by using a cell counting plate, and diluting the cell suspension by adding a cell culture medium to ensure that the cell density reaches 4 multiplied by 10 5 Cells were then split into 24-well plates and 3 wells of cells were counted daily for 10 days. Cell growth curves and specific growth rate curves were plotted according to cell numbers.
The results are shown in fig. 6, where all cells were grown with a latency period, an exponential growth phase, and a lag phase. Under the condition of inoculating the same cell density, the CHO-HS3ST5-OE-1 recombinant cells are similar to the growth curve of CHO-K1 cells, and the cell numbers reach a peak value on the 6 th day. FIG. 7 shows that the specific growth rates of CHO-HS3ST5-OE-1 recombinant cells and CHO-K1 cells are similar and negative after day 6, consistent with growth curve results showing that stable expression of HS3ST5 does not substantially affect the growth performance of CHO-K1 cells.
Example 4
Analysis of expression stability of HS3ST5 protein in recombinant CHO-K1 cell stably expressing HS3ST5
To analyze the stability of HS3ST5 protein expression in recombinant cell lines, cells of passage 5 and 20 were harvested to determine whether the expression of HS3ST5 protein was stable.
The stability of HS3ST5 protein expression during passages of CHO-HS3ST5-OE-1 recombinant cells was analyzed by WesternBlot. As shown in fig. 8, the levels of HS3ST5 protein expressed by the 5 th and 20 th generation recombinant cell lines were not significantly different, indicating that HS3ST5 protein was stably expressed in the recombinant cell lines.
Example 5
Effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV adsorption
The effect of recombinant CHO-K1 cells on FMDV adsorption was evaluated by detecting FMDV 3D mRNA expression levels. Counting cells, adding 4 ℃ precooled FM DV with an infectious dose (MOI) of 1, standing for 2 hours at 4 ℃ to enable the FMDV to fully adsorb the cells, washing off unadsorbed FMDV by using 4 ℃ precooled PBS buffer solution, then adding RNA lysate, extracting RNA after collecting samples, and detecting the 3D mRNA level adsorbed to the cell surface FMDV by using an RT-qPCR method. Wherein, the detection primer used by RT-qPCR refers to the RT-qPCR primer in published literature, the upstream primer for amplifying FMDV 3D gene is 5'-ACTGGGTTTTACAAACCTGTGA-3' (SEQ ID NO: 6), and the downstream primer is 5'-GCGAGTCCTGCCACGACGGA-3' (SEQ ID NO: 7); the upstream primer of the amplified reference gene GAPDH gene was 5'-CAAGAAGGTGGTGAAGCA-3' (SEQ ID NO: 8) and the downstream primer was 5'-AAGTGGAAGAGTGAGTGTC-3' (SEQ ID NO: 9). The reverse transcription system of the RT-qPCR method is shown in Table 2, and the reverse transcription procedure is 15min at 37℃and 5s at 85 ℃. The amplification system of the RT-qPCR method is shown in Table 3, and the amplification reaction procedure is 30s pre-denaturation at 95 ℃;95 ℃ for 5 seconds, 60 ℃ for 30 seconds, 72 ℃ for 30 seconds, 40 cycles; 95℃15s,60℃1min,95℃30s,60℃15s. The relative mRNA copy number of FMDV 3D was calculated using the ΔΔct method.
TABLE 2 RT-qPCR reverse transcription System of FMDV 3D
Table 3 amplification System for RT-qPCR of FMDV 3D
The effect of CHO-HS3ST5-OE-1 recombinant cells on FMDV internalization was examined using an RT-qPCR assay. As shown in FIG. 9, the FMDV mRNA level in recombinant CHO-K1 cells stably expressing HS3ST5 was significantly increased relative to normal CHO-K1 cells, and the viral mRNA level in CHO-HS3ST5-OE-1 was 4.3 times higher than in control cells, indicating that recombinant CHO-K1 cells stably expressing HS3ST5 facilitated adsorption of FMDV.
Example 6
Effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV internalization
The test procedure for detecting internalization of FMDV in recombinant CHO-K1 cells is as follows: earlier procedure with adsorption experiments, 4 ℃ pre-chilled PBS buffer washed off unadsorbed FMDV, cells were placed at 37 ℃ for 0.5h, cells were treated with PBS buffer at pH 2.5 for 5min to inactivate adsorbed but unadsorbed virus, then RNA lysate was added, RNA was extracted after taking samples, and 3D mRNA levels of internalized FMDV were detected using RT-qPCR method described in example 5.
The effect of CHO-HS3ST5-OE-1 recombinant cells on FMDV internalization was examined using an RT-qPCR assay. As shown in FIG. 10, the FMDV mRNA level in recombinant CHO-K1 cells stably expressing HS3ST5 was significantly increased relative to normal CHO-K1 cells, and the viral mRNA level in CHO-HS3ST5-OE-1 was 5.9-fold higher than in control cells, indicating that recombinant CHO-K1 cells stably expressing HS3ST5 are beneficial for FMDV internalization.
Example 7
Effect of recombinant CHO-K1 cells stably expressing HS3ST5 on FMDV Gene replication
Recombinant CHO-K1 cells were inoculated with a virus having MOI of 1, placed in an incubator at 37℃and samples were harvested by adding RNA lysates after 5, 10, 15, 20, 25, 30, 35 and 40 hours, respectively. RNA was extracted and 3D mRNA levels of FMDV were detected by RT-qPCR.
The effect of CHO-HS3ST5-OE-1 recombinant cells on FMDV gene replication was detected using an RT-qPCR assay. As shown in fig. 11, FMDV mRNA levels were significantly increased in recombinant CHO-K1 cells stably expressing HS3ST5 compared to normal CHO-K1 cells for all time point samples. FMDV mRNA levels in CHO-HS3ST5-OE-1 recombinant cells and normal CHO-K1 cells peaked in 25h samples, when the FMDV mRNA levels in CHO-HS3ST5-OE-1 were 2.9-fold higher than in control cells. The above results demonstrate that recombinant CHO-K1 cells stably expressing HS3ST5 enhance gene replication of FMDV.
Example 8
Effect of recombinant CHO-K1 cells stably expressing HS3ST5 on the proliferation of sub-generation FMDV
Recombinant CHO-K1 cells stably expressing HS3ST5 were infected with FMDV and progeny virus quantified by plaque formation assay. FMDV with MOI of 1 was inoculated with recombinant CHO-K1 cells and normal CHO-K1 cells, and samples were collected after 4, 8, 12 and 16h in a 37℃incubator, respectively, and the plaque assay was performed to determine FMDV Plaque Forming Units (PFU) in the samples.
Samples of FMDV infected recombinant CHO-K1 cells and normal CHO-K1 cells were collected for different durations and progeny FMDV was quantified by plaque formation assays. As shown in FIG. 12, the number of plaques on recombinant CHO-K1 cells stably expressing HS3ST5 was significantly increased in all time point samples compared to normal CHO-K1 cells. The number of FMDV plaques peaked 25h after viral infection, at which point the number of plaques in CHO-HS3ST5-OE-1 was 2.2 times that in control cells. The above results indicate that recombinant CHO-K1 cells stably expressing HS3ST5 enhance the proliferation of progeny FMDV.
From the above examples, it was found that the HS3ST5 fragment was obtained by RT-PCR amplification, cloned into a pLOV-CMV-EGFP vector to construct a recombinant lentiviral plasmid, and then the recombinant lentiviral plasmid pLOV-EGFP-CHO-HS3ST5 and helper plasmids pMD2.G and psPAX2 were transfected into HEK-293T cells by liposome transfection technique, the lentivirus packaged by HEK-293T cells was harvested, and then CHO-K1 cells were infected, puromycin was continuously screened for 10 days to obtain a recombinant CHO-K1 cell line expressing HS3ST5, and a monoclonal recombinant CHO-K1 cell line stably expressing HS3ST5 was screened by limiting dilution and green fluorescence intensity observation. The HS3ST1, 2, 4, 5 and 6 genes in CHO-K1 cells were reported to be transcribed but not express the protein, and established recombinant CHO-K1 cells successfully expressed the HS3ST5 protein, indicating that CHO-K1 cells themselves have the ability to express the HS3ST5 protein, but may be under the control of a variety of factors, resulting in the silencing or too low level of endogenous HS3ST5 protein expression in CHO-K1 cells to be detected. The genetic stability of the HS3ST5 protein expression in the 5 th and 15 th generation recombinant CHO-K1 cells is also detected, and the screened positive monoclonal recombinant CHO-K1 cells can continuously and stably express the HS3ST5 protein, which indicates that the HS3ST5 gene is stably integrated into the CHO-K1 cells, and also indicates that the obtained target cells have good genetic stability. In addition, the present study also analyzed the growth rate and specific growth rate of recombinant CHO-K1 cells, and the results showed that recombinant cells and normal cells have similar growth trends, indicating that overexpression of HS3ST5 did not affect the growth characteristics of CHO-K1 cells. Overall, a recombinant CHO-K1 cell line stably expressing HS3ST5 was successfully established.
And (3) by using the established recombinant CHO-K1 cells stably expressing HS3ST5 and taking FDMV as a virus model, determining the adsorption, internalization, gene replication and progeny virus level of FMDV on the recombinant CHO-K1 cells, and finally evaluating the influence of the recombinant cells on the replication capacity of the FMDV. The results show that recombinant CHO-K1 cells stably expressing HS3ST5 promote adsorption, internalization, gene replication and progeny virus production of FMDV. In general, recombinant CHO-K1 cells stably expressing HS3ST5 increased the replication capacity of FMDV. It can also be seen that HS3ST5 affects the overall infection process of FMDV by affecting the adsorption of FMDV to cells. This is consistent with literature reports that HS3ST modified HS is capable of promoting adsorption levels of viruses such as CMV and HSV-1 and thus promote viral infection.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Use of an agent that overexpresses the HS3ST5 gene for promoting viral infection.
2. The use according to claim 1, wherein the nucleotide sequence of the HS3ST5 gene is shown in SEQ ID No. 1.
3. The use according to claim 1, wherein the agent that overexpresses the HS3ST5 gene comprises a recombinant vector that overexpresses the HS3ST5 gene.
4. The use of claim 1, wherein said increasing viral infectivity comprises increasing viral adsorption and the ability to invade host cells, increasing viral gene replication and progeny viral replication levels.
5. The use according to any one of claims 1 to 4, wherein the virus comprises a virus which invades cells using HS receptors.
6. The use according to claim 5, wherein the viruses that invade cells using HS receptors comprise one or more of foot-and-mouth disease virus, severe acute respiratory syndrome coronavirus and herpes simplex virus.
7. A recombinant CHO-K1 cell line stably expressing HS3ST5, wherein the recombinant CHO-K1 cell line overexpresses HS3ST5 protein.
8. The method for constructing a recombinant CHO-K1 cell line stably expressing HS3ST5 according to claim 7, comprising the steps of:
1) Constructing a recombinant lentiviral plasmid over-expressing HS3ST5 gene;
2) Co-transfecting the recombinant lentiviral plasmid and the auxiliary plasmid of the over-expressed HS3ST5 gene in the step 1) into cells to rescue to obtain the over-expressed HS3ST5 recombinant lentivirus;
3) The recombinant lentivirus obtained by rescue is infected with CHO-K1 cells, and a recombinant CHO-K1 cell line which stably expresses HS3ST5 is obtained by screening.
9. The construction method according to claim 8, wherein the construction method in step 1) uses a primer containing Xba I and Not I cleavage sites to amplify the HS3ST5 gene, and the obtained amplified fragment and lentiviral vector pLOV-CMV-EGFP are respectively digested by Xba I and Not I, and the digested fragment and linearization vector are connected, and the recombinant lentiviral plasmid over-expressing the HS3ST5 gene is obtained.
10. Use of the recombinant CHO-K1 cell line stably expressing HS3ST5 according to claim 7 or the recombinant CHO-K1 cell line stably expressing HS3ST5 obtained by the construction method according to claim 8 or 9 for the preparation of a virus infected cell model.
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