CN112553164A

CN112553164A - Genetically modified stem cell for treating COVID-19

Info

Publication number: CN112553164A
Application number: CN202011383918.0A
Authority: CN
Inventors: 翁炳焕
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-11-22
Filing date: 2020-11-22
Publication date: 2021-03-26

Abstract

A gene modified stem cell for treating COVID-19 is characterized in that mesenchymal stem cells or amniotic fibroblasts are obtained from a stem cell sample bank or a sample remained after a daily experiment, stem cells are screened and induced, then a susceptibility gene ACE2 of a new coronavirus is inserted into the DNA of an immortalized stem cell transfected by SV40LT and/or hTERT, and the susceptible stem cell which is easily infected by the new coronavirus is prepared; and further inserting the RNA interference sequence shRNA of the new coronavirus M, N, E and/or S gene into the prepared susceptible stem cell DNA to construct a genetically modified stem cell which can absorb the new coronavirus into the stem cell through ACE2, can target and interfere the replication of the new coronavirus in the stem cell through the shRNA, and can be amplified in vitro through unlimited passage.

Description

Genetically modified stem cell for treating COVID-19

Technical Field

The invention relates to a genetically modified stem cell for treating COVID-19, belonging to the technology of infectious disease control in the field of biomedicine.

Background

The novel coronavirus (SARS-CoV-2) has posed a serious threat to global public health. The main structure of SARS-CoV-2 comprises single-stranded positive-strand nucleic acid (ssRNA), spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). Wherein the S protein is cleaved by host protease into an N-terminal S1 subunit and a C-terminal S2 subunit in the infection process, the S1 subunit is composed of an N-terminal structural domain (S1-NTD) and a receptor binding domain (S1-RBD), the S1-RBD is responsible for recognizing and binding a host cell surface receptor angiotensin converting enzyme 2(ACE2), and the S2 subunit mediates fusion between a virus envelope and a host cell membrane, so that the virus enters cells to cause infection. The N protein is essential for RNA synthesis, plays a crucial role in viral assembly and RNA transcription, and is also involved in host cell response to viral infection. The M and E proteins play an important role in viral assembly.

At present, stem cells for clinical treatment mainly focus on Mesenchymal Stem Cells (MSCs) and natural killer cells (NK) at home and abroad, and the MSCs are most widely used. MSCs are derived from mesoderm in early development, belong to pluripotent stem cells, can migrate to the exact part of injury, can be directionally differentiated into various cell lines such as lung histiocyte and capillary endothelial cells, generate various cytokines, secrete a large amount of exosomes and vesicles containing miRNA, treat lung injury by influencing signal pathways such as PI3K/AKT and NF-kB, and repair damaged organs by mechanisms such as immunity regulation, fibrosis resistance, inflammatory factor storm inhibition and the like. The stem cells have very strong antiviral ability, can survive and play a role in local viral infection, preliminarily show the safety and effectiveness of the MSCs in the treatment of severe new coronary pneumonia, and have good clinical application prospect. However, no reports are found in the literature on the treatment of COVID with functionally modified stem cells.

Usually, the mesenchymal stem cells will age or die after in vitro passage to 15-30 generations, while the cell line transfected by the simian virus 40 large T antigen gene (SV40LT) can be cultured in vitro for more than 350 generations, and can basically keep the differentiation phenotype and biological characteristics of the original cells, and the mesenchymal stem cells are widely applied to the immortalization of human liver cells, vascular striation marginal cells, cartilage stem cells and the like. This provides the basis for the improvement of the culture life of the stem cells in vitro and the industrial expansion. The literature reports that SARS-CoV replicates in ACE2 transfected 293T cells but not in mock transfected 293T cells. It is now reported that SARS-CoV-2 also infects host cells via the ACE2 receptor. In the traditional concept, SARS-CoV-2 is not expected to infect body cells and therapeutic stem cells through ACE2 receptor, but the invention constructs a recombinant vector expressing ACE2 in a reverse method, transfects the stem cells to enhance the function of the stem cells expressing ACE2, thereby enhancing the susceptibility of the stem cells to SARS-CoV-2, enables the stem cells to compete with host cells for inhaling more new coronavirus in treatment so as to reduce the infection chance of normal tissues of the body, and then inhibits and kills the new coronavirus introduced into the stem cells by ACE2 through RNA interference genes artificially assembled on the DNA of the stem cells.

RNA interference (RNAi) refers to the antiviral effect of specifically silencing a foreign gene. When the exogenous target gene is integrated into the host cell genome, the exogenous target gene can utilize the host cell to transcribe dsRNA, the dsRNA is cut into a plurality of small-fragment siRNAs (about 21-23 bp) with specific length and structure by endonuclease (Dicer) in host cytoplasm, the siRNAs are melted into a sense strand and an antisense strand under the action of intracellular RNA helicase, and the antisense siRNAs are combined with the endonuclease, the exonuclease and the helicase in vivo to form an RNA-induced gene silencing complex (RISC). When the exogenous gene invades the host cell, the RISC is specifically combined with the homologous sequence of the mRNA expressed by the exogenous gene, the homologous mRNA is cut at the combination part, and the cut broken mRNA is immediately degraded, thereby inducing the degradation of the host cell aiming at the exogenous mRNA. The siRNA can not only guide RISC to cut homologous single-stranded mRNA, but also can be used as a primer to be combined with target RNA and synthesize new dsRNA under the action of RNA polymerase, the newly synthesized dsRNA is cut into a large amount of secondary siRNA by Dicer, and then RISC is formed to play a role, so that the function of RNAi is further amplified, and finally the exogenous target mRNA is completely degraded. However, no report is found for preparing a new coronavirus vaccine by taking artificially constructed stem cells with RNA interference function as vectors.

Disclosure of Invention

The present inventors have made the present invention based on the above-mentioned problems of the stem cells for current therapy.

The invention aims to prepare a genetically modified stem cell which is artificially assembled with an immortalized gene, a new coronavirus susceptibility gene and an RNA interference gene.

The purpose of the invention is implemented by the following technical scheme:

first, mesenchymal stem cells are obtained. For example, mesenchymal stem cells and/or amniotic fluid fibroblasts are obtained from a stem cell sample bank or a sample remaining after a daily experiment, and screened or induced into lung stem cells.

Further, stem cells are assembled with immortalizing genes. For example, a recombinant vector carrying the hTERT and/or SV40LT is constructed, stem cells are transfected, and the DNA of the stem cells is integrated with the hTERT and/or SV40LT genes, so that a new function capable of permanent survival and unlimited passage is obtained, and a lung stem cell line can be obtained through identification or induction.

Further, stem cells are assembled with a new coronavirus susceptibility gene. For example, angiotensin converting enzyme II (ACE2) gene was ligated into lentiviral expression vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro or pGC-FU to construct recombinant plasmid pHBLV-OE-ACE2 or pGC-FU-ACE2, respectively, recombinant plasmid pHBLV-OE-ACE2 and packaging plasmids (psPAX2 and pMD2G) or recombinant plasmid pGC-FU-ACE2 and packaging plasmids (pHelper1.0 and pHelper2.0) were co-transfected into 293FT cells, recombinant lentivirus OE-ACE2 or FU-ACE2 carrying ACE2 was packaged, and recombinant lentivirus was transfected into stem cells to integrate ACE2 gene into stem cell DNA. Thereby enhancing the virus tropism of the stem cells, namely being more easy to compete with host cells for sucking nCoV and carrying out RNA interference in the stem cells to kill the nCoV.

Further, stem cells are assembled with a novel coronavirus RNA interference gene. For example, the targeting interfering sequence siRNA of a new coronavirus (nCoV) is designed and preferred, an shRNA template is synthesized, the siRNA template is connected to a lentiviral vector pHBLV or LV3 to construct a recombinant plasmid pHBLV-nCoV-N-shRNA or LV3-nCoV-N-shRNA, the recombinant plasmid pHBLV-nCoV-N-shRNA and a packaging plasmid (pHBLV, a psPAX2 vector and a pMD2G vector) or the recombinant plasmid LV3-nCoV-N-shRNA and a packaging plasmid (pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5) are co-transfected into 293FT cells to package lentiviruses carrying the shRNA, the lentiviruses are transfected into stem cells, the shRNA gene is integrated into the stem cell DNA, and thereby obtaining a new function capable of interfering the replication of nCoV in the stem cells.

Further, the genetically modified stem cell which is artificially assembled with the immortalization gene, the susceptibility gene and the RNA interference gene and has the natural treatment function of the stem cell, the artificially improved immortalization function, the susceptibility function of the new coronavirus and the RNA interference function is prepared.

The invention has the beneficial effects that:

the tissue cells which are abandoned originally are transformed into genetically modified stem cells with a plurality of new functions.

The genetically modified stem cell solves the problems of ultralow temperature permanent storage, renewable utilization and commercialization requiring infinite amplification due to the assembly of immortalized genes (SV40LT and/or hTERT).

The genetically modified stem cell is assembled with RNA interference gene of new coronavirus, so that the virus is more easily inhibited from replicating in the stem cell, and the stem cell which is originally easy to kill virus is modified into the genetically modified stem cell which is easy to kill virus.

Genetically modified stem cells assemble a new coronavirus susceptibility gene (ACE2) on the basis of assembling an RNA interference gene, and are more likely to compete with host cells to take viruses into the stem cells and perform RNA interference in the stem cells, so that the infection chances of the host cells are reduced. The application method of the ACE2 overcomes the traditional prejudice that therapeutic stem cells are generally worried about being infected by viruses, and the ACE2 gene assembled in the genetically modified stem cells can express ACE2 protein, the protein can stimulate a host to produce ACE2 antibody, and the ACE2 antibody can prevent new coronavirus from infecting new host cells due to the fact that the binding of the new coronavirus and an ACE2 receptor can be inhibited.

Meanwhile, the gene modified stem cell assembled with the ACE2 gene and the RNA interference gene absorbs viruses into the stem cell, and inhibits the viruses from being replicated in the stem cell to play the roles of introducing the viruses into the cell and killing the viruses in the cell, so that the antiviral reaction with the tissue damage function is transferred from the host tissue to the stem cell, and the damage of the host tissue caused by the antiviral reaction can be reduced.

Drawings

FIG. 1 is a clone of immortalized cells screened with G418 and puromycin according to the present invention.

FIG. 2 is a subculture map of a cell clone transfected with SV40LT and hTERT according to the present invention.

FIG. 3 is the cell growth status of stem cells of recombinant ACE2 co-cultured with isolated virus for 72 hours.

FIG. 4 is the cell growth status of stem cells of recombinant ACE2 and shRNA co-cultured with isolated virus for 72 hours.

In FIG. 1, since immortalized cells transfected with SV40LT and hTERT survived without being killed by G418 and puromycin, surviving single cells grew to form cell clones.

In FIG. 2, cells transfected with SV40LT and hTERT grew adherent in fusiform during passage and grew vigorously.

In FIG. 3, the cells are seen to be round, floating and dead, indicating that the expression of ACE2 gene in the cells is favorable for the virus to be absorbed into the cells and to multiply in the cells, resulting in faster cell death.

In FIG. 4, it can be seen that the cells are still growing fusiform adherent, indicating that the expression of shRNA interferes with intracellular replication of the virus, allowing stem cells that have recombined ACE2 and shRNA to take the virus into and kill it intracellularly.

Detailed Description

The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings, but these exemplary descriptions should not be construed as limiting the scope of the present invention as defined in the appended claims.

EXAMPLE one preparation of genetically modified Stem cells

1. Collection of Mesenchymal Stem Cells (MSC)

1.1. Collection of amniotic fluid mesenchymal stem cells

Collecting amniotic fluid cells of the pregnant woman to be detected according to the prenatal diagnosis process, performing cell culture and prenatal diagnosis, and screening out fusiform amniotic fluid cells or mesenchymal stem cells growing adherently from the remaining amniotic fluid cells under an inverted microscope.

1.2. Collection of umbilical cord mesenchymal stem cells

Taking out the frozen umbilical cord from liquid nitrogen tank, thawing rapidly in 37 deg.C water bath, cleaning with sterile PBS, removing residual blood stain, and cutting into size of about 1mm³The tissue block (2) is placed in a culture dish paved with fetal calf serum, placed at 37 ℃ for 6 hours, added with 10% fetal calf serum DMEM medium, and 5% CO at 37 DEG C₂Culturing in an incubator, observing the growth condition of cells around adherent tissues, and digesting and passaging by pancreatin when the cells reach 80-90% fusion degree to obtain the mesenchymal stem cells.

1.3. Collection of mesenchymal Stem cells

Aseptically taking lung tissue of abortion fetus, mechanically dispersing, digesting with 0.25% trypsin, filtering with gauze with pore diameter of 100 μm, centrifuging at 1000 r/min5min, remove the supernatant and add DMEM medium (0.1umol beta-mercaptoethanol, 100UI/mL streptomycin, 10% fetal bovine serum). At 37 deg.C, 5% CO₂Culturing under the condition. And changing the solution after 45min to remove the cells which are not attached to the wall, and changing the solution every 48 h. After 80% cell confluence, 0.25% trypsin digestion was used for passage.

2. Immortalization performance improvement of mesenchymal stem cells

2.1 construction of recombinant vectors for hTERT/pLPCX and SV40LT/pLXSN

Construction of SV40LT/pLXSN

Performing PCR amplification on SV40LT high-fidelity long fragments by using SV40DNA (strain 766) as a template, an upstream primer of 5'-GCCCAGGATCCTTAACAACAACAACAAT-3' and a downstream primer of 5'-ACGCTGAATTCCCTCTGAGCTAT-3'; the PCR product of SV40LT and the pLXSN retrovirus vector are subjected to enzyme digestion, connection, transformation, screening and sequencing verification by EcoR I/BamH I to obtain the SV40 LT/pLXSN-containing recombinant retrovirus vector.

2.1.2. construction of hTERT/pLPCX

Cutting PIRES2-EGFP-hTERT plasmid with EcoR I to obtain hTERT DNA fragment, dephosphorylating, subcloning to pLPCX retrovirus vector EcoR I site, connecting with T4DNA ligase at 22 deg.C, transforming to competent E.coli DH5-alpha E, culturing overnight at 37 deg.C, selecting colorless colony for inoculation, purifying recombinant plasmid, performing enzyme cutting and sequencing identification, expanding and culturing hTERT-containing E.coli, and purifying pLPCX-hTERT recombinant clone with Endotoxin-Free plasmid.

2.2. Combined transfection of SV40LT and hTERT into mesenchymal stem cells

Mesenchymal cells to be transfected were matched to approximately 8X 10⁵Inoculating in single cell concentration, and placing in 5% CO₂And culturing at 37 ℃, infecting with recombinant retrovirus containing SV40LT gene (Polybrene concentration is 8ug/mL) after 24h, screening with 500ug/mL G418 for 4 weeks after 1 week, infecting with recombinant retrovirus containing hTERT gene (Polybrene concentration is 8ug/mL) after cell cloning appears, and screening with puromycin 2ug/mL after 1 week to obtain cell clone (see figure 1).

2.3. Identification of immortalized mesenchymal stem cells

2.3.1. Biological characterization of immortalized stem cells

Includes that the cell is spindle-shaped and fibroid. ② white bands appear at the relative molecular mass of 120000 and 93000 respectively by Western detection. ③ the growth curve of the cell line is typical of the "S" growth characteristics. And fourthly, the chromosome karyotype of the cell line is diploid. The cell line can not grow in soft agar. Sixthly, the nude mouse has negative tumorigenicity test.

2.3.2. Surface molecule detection of immortalized stem cells

Detecting cell membrane surface molecules by using a flow cytometer, wherein positive molecules comprise CD73-APC, CD90-FITC, CD44-PE and CD 105-Cy5.5; negative molecules include CD11b-PE, CD19-PE, CD34-PE, CD45-PE, HLA-DR-PE. Through identification, the immortalized mesenchymal stem cells which can be permanently passaged in vitro are obtained. FIG. 2 shows stem cells passed to passage 35.

2.3.3. Identification of Lung Stem cell lines

The human lung stem cell line needs to meet the following requirements: observing with a phase contrast microscope: growing in shuttle shape and arranging in vortex or barrier shape. Detecting by a flow cytometer: cell surface markers CD45, CD11 a, CD14, CD90, CD34, CD71, CD25, CD105, CD117, CD166 and CD44 were positive. And thirdly, confocal technology detection: keratin expression is negative, and four sternness related factors c-Myc, Oct4, Nanog and Nestin are positive. Fourthly, indirect immunofluorescence detection: vimentin (vimentin), collagen type III (collagen III), Fibronectin (FN) expression were positive, while Surfactant protein C precursor (pro-Surfactant protein C, proSP-C), von Willebrand factor (vWF), and alpha smooth muscle actin (alpha-SMA) expression were negative.

3. Assembly of a novel coronavirus susceptibility gene (ACE2) for immortalized stem cells

3.1. Construction and identification of lentiviral expression vector pHBLV-ACE2-OE or pGC-FU-ACE2

ACE2 in plasmid pc-DNA3.1-hygro (+) -mACE2 (or RNA extracted from lung tissue cells, transcribed as cDNA, and then ACE2 PCR-amplified) was amplified by PCR. Designing and synthesizing a PCR primer sequence according to the mRNA sequence of human ACE2 in GenBank, wherein an outer end primer amplified by human ACE 2: f1(F out) 5'-GAT GGA GTA CCG ACT GGA GTC-3', R1(Rout) 5'-CTA ATA TCG ATG GAG GCA TAA-3', product 547 bp; an inner end primer: f2(F in) 5'-GAG GAG GAT GTG CGA GTG GCT A-3', R2(R in) 5'-CCA ACC ACT ATC ACT CCC ATC A-3', and a product of 269 bp. The amplification primer sequence of the human beta-actin is as follows: F5'-GCT CGT CGT CGA CAA CGG CTC-3', R5'-CAA ACA TGA TCT GGGTCATCTTCT-3', product 353 bp. PCR conditions were as follows: 94 deg.C for 5min, then 94 deg.C for 30s denaturation, 55 deg.C for 30s annealing, 68 deg.C for 5min, circulating for 30 times, and finally extending at 68 deg.C for 10 min.

The vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro and the PCR product are respectively cut by BamH I and EcoRI, or pGC-F and the PCR product are respectively cut by AgeI and EcoRI, agarose electrophoresis is used for purification, linearized vector DNA, the PCR product recovered by enzyme cutting, T4 phage DNA ligase and buffer thereof and ddH20 are connected at 16 ℃ overnight, the connecting liquid is transformed into competent cells, and positive clones are identified by PCR and sequencing. The transformed bacteria are amplified by LB culture solution, pHBLV-ACE2-OE or pGC-FU-ACE2 is extracted by a plasmid extraction kit, 293FT cells are transfected by Lipofectamine 2000, and expression of ACE2 genes is detected by Western-Blot after 72h of transfection.

3.2. Packages carrying ACE2 lentivirus and titer determination thereof

The recombinant lentivirus (OE-ACE2) or Lentiviral-ACE carrying ACE2 gene was obtained by co-transfecting 293T cells with lentivirus expression vectors (pHBLV-OE-ACE2), packaging plasmids (psPAX2 vector and pMD2G) or pGC-FU-ACE2 and packaging plasmids (pHelper1.0 and pHelper2.0) as described in Invitmgen Upofectamine 2000, respectively.

At the same time, another set of 293T cells was co-transfected with pHBLV empty vector (containing GFP gene), packaging plasmids (psPAX2 vector and pMD2G), and the empty vector (NC-GFP) carrying only GFP gene was obtained as a control.

And 8h after transfection, replacing the medium with a complete medium, culturing for 48h, collecting cell supernatant rich in lentivirus, centrifuging for 10min at 4 ℃ at 4000 g, filtering the supernatant by using a 0.45-micron filter, removing cell debris, and centrifuging to obtain the lentivirus with high titer.

Infecting 293T cells with the virus stock solution to be identified, extracting RNA after 4d, and determining the virus titer of the lentivirus to be detected by using a Real-Time PCR method.

3.3. Recombinant lentivirus transfection of immortalized stem cells

Taking immortalized stem cells at a rate of 1X 10 per well⁶Inoculating the cells at a density of 6-well plates, dividing into a target gene over-expression (OE-ACE2) group, an empty vector (NC-GFP) control group and a blank group, wherein each group has 2 wells, adding a lentiviral stock solution diluted by 5 times into a lentiviral group when the cells are converged to 30%, adding an empty vector stock solution diluted by 5 times into an empty vector group, culturing for 24h, replacing a 10% FBS DMEM solution, and adding puromycin (2.50 mu g. mL) with an optimal screening concentration^-1) Puromycin concentration was maintained and fluid changes were performed every other day until the blank cells were completely dead and selection was complete (Lentiviral-ACE transfected cells were selected with ampicillin). The cells which are not killed by puromycin are stem cell lines which are transfected by recombinant lentiviruses and have integrated ACE2 gene on DNA, and are called recombinant ACE2 stem cells.

3.4.ACE2 gene detection of recombinant ACE2 stem cells

RT-PCR detection of mRNA transcript levels of the ACE2 Gene

Taking ACE2 stem cells, and culturing at a ratio of 1 × 10⁶And inoculating the cells in a 6-well plate at a density, culturing for 4 days, observing the expression of fluorescence by using a fluorescence microscope, and detecting the transcription level of the ACE2 gene in ACE2 stem cells by using RT-PCR. The primer sequence of the RT-PCR amplification ACE2 is as follows: upstream of ACE 2: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3', respectively; downstream: EF 1-Rn: 5'-GCCAGTACACGACATCACTT-3', respectively; the upstream and downstream of beta-actin are respectively: 5'-TGGACTTCGAGCAAGAGATGG-3', 5'-ATCTCCTTCTGCATCCTGTCG-3'. RT-PCR detected high level transcription of the ACE2 gene in OE-ACE2 group, showing 1 additional 409bp mRNA band transcribed from ACE2 compared to control. And through comparison and statistical analysis of ACE2/B-actin integrated optical density value (IOD), the mRNA expression quantity of genetically modified stem cells carrying ACE2 gene is obviously increased (p is less than 0.01) (the normal cell group is 0.234 +/-0.101, the empty vector control group is 0.247 +/-0.098, and the ACE2 stem cell group is 0.682 +/-0.118), and the suggestion is that the mRNA expression quantity is obviously increasedIncrease ACE2 transcription.

Western-Blot detection of protein expression levels of ACE2 stem cells

Extracting proteins of cell lysates of each group, quantifying the proteins by a BAC method, and arranging 3 random multiple holes in each group. The sample proteins were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, primary antibody was anti-VDR diluted 1: 400, visualized using ECL chemiluminescence kit, and the relative gray values of the protein bands determined using a gel image analysis system. Western-Blot detected that the ACE2 gene expression of ACE2 stem cell group is obviously increased, and shows 1 very obvious protein expression band (ACE2-90kDa) expressed by ACE2, while the normal cell group and the empty vector group only have 1 same beta-actin (42kDa) band.

4. Novel coronavirus RNA interference gene assembly of recombinant ACE2 stem cells

4.1. Design of siRNA Gene of novel coronavirus

By Ambion corporation (http：//www.ambion.com/techlib/misc/siRNAtools.html) The shRNA online design software of (2) screens a plurality of siRNA alternative sequences from the conserved sequences of SARS-CoV-2 (NC-045512.2 strain ORFlab, 3' UTR, S, E, M, N).

Based on the database of human genome, Tm value of RNA binding and the result of specific alignment, 3 conservative siRNA sequences without homology with human genome are respectively preferred (Table 1A), and 1 irrelevant siRNA sequence is designed as negative control (NC: 5'-TTCTCCGAACGTGTCACGTAA-3').

TABLE 1A siRNA candidate sequence of the S, E, M, N gene of NC-045512.2 strain neocoronavirus

4.2. Synthesis of shRNA templates

After the target sequence has been determined, the vector pHBLV-U6-MCS-CMV-Z is subjected to a lentivirus interferenceThe polyclonal restriction enzyme cutting site of sGreen-PGK-PURO is designed with shRNA template, each template is composed of two most complementary single-stranded DNA with length of 52-60nt, the 3' end of oligonucleotide single-stranded DNA has 2-5U-shaped protrusions, including sense sequence of target sequence, loop sequence, antisense sequence of target sequence, transcription termination signal and cohesive end sequence after restriction enzyme cutting, and after annealing and complementation, a DNA double-strand with cohesive ends of BamH I and ECORI restriction enzyme cutting site can be formed (for example, pSilencer4.1.CMV. neo, BamH I and Hind III are used). As shown in Table 1B, "italics" indicates the cleavage site, "bold" indicates the stem-loop structure, ")"Underlining"denotes the sense strand.

Table 1B Synthesis of shRNA templates corresponding to S, E, M, N Gene with siRNA in Table 1A

4.3. Construction of Lentiviral interference vectors

Taking a targeting interference sequence (nCoV-N-siRNA-1/2/3) of a new coronavirus N gene as an example, a T4 ligase is used for connecting a synthesized shRNA template with a linearized lentiviral vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO to construct a lentiviral interference vector pHBLV-nCoV-N-shRNA1/2/3, and the transformation competence E.coli DH5 alpha.

4.4. Identification of lentiviral interference vectors

Transformation of lentiviral interference vectors to competent E.coli DH 5. alpha.10. mu.L, plated in solution containing 50. mu.g.mL^-1And (3) performing resistance culture on an LB (lysogeny broth) plate of puromycin, screening out positive clones, extracting plasmids, performing enzyme digestion or sequencing identification by using PCR (polymerase chain reaction), BamH I and ECORI (isocoryl chloride), culturing the positive clones with correct sequencing, and extracting the plasmids according to a plasmid extraction kit. And observing whether the 3 targeted nCoV-N-siRNA1/2/3 interference genes in the recombinant lentiviral interference vector are completely consistent with the designed interference sequence.

4.5. Packaging with shRNA lentivirus

293FT cells in logarithmic growth phase were taken at 5X 10⁶cell·mL^-1The recombinant plasmid and the lentivirus packaging plasmid (consisting of three plasmids of pHBLV, psPAX2 vector and pMD2G vector) are added into a culture bottle, the recombinant plasmid and the lentivirus packaging plasmid (consisting of 4 mug of pHBLV, psPAX2 vector and pMD2G vector) are added into the culture bottle, the culture bottle is cotransfected with Lipofectamine200 liposome, the culture medium is changed into a complete culture medium after 8 hours, the culture is continued for 48 hours, and then the supernatant rich in lentivirus is collected, centrifuged, filtered, subpackaged and stored at-80 ℃.

4.6. Detection of lentivirus titre

Wells dilution method for counting fluorescent cells: taking 10 μ L of lentivirus stock solution, diluting with 10 times of 10% FBS DMEM culture solution to obtain 3-5 gradients, and adding 293FT cells at a rate of 3 × 10 per well⁴The density of each cell was seeded in 96-well plates at 37 ℃ with 5% CO₂After 24 hours of incubation, 150. mu.L of DMEM medium was replaced with 10% FBS per well and incubation was continued for 48 hours, and the virus titer was calculated by counting the fluorescent cells using a fluorescence microscope. As a result, after 293FT cells were transfected with the recombinant plasmid pHBLV-nCoV-N-shRNA (LV3-nCoV-N-shRNA), the plasmid was packaged into lentiviral particles, and green fluorescence was observed in the 293FT cells under a fluorescence microscope. Second, Real time quantitative PCR method determination: 293FT cells were cultured in 10% FBS DMEM, infected with lentivirus stock to be assayed, RNA was extracted according to the TRIZOL protocol of Invitrogen, and the concentration of the extracted RNA was determined by RT-qPCR.

4.7. Lentiviral transfection of recombinant ACE2 stem cells

At a rate of 1X 10 per hole⁶According to the cell density, ACE2 stem cells with good growth state are inoculated to a 6-well plate, and when the cells are fused to 30%, the cells of a test group are respectively added with 5-fold diluted lentivirus stock solution and are respectively named as pHBLV-nCoV-N-shRNA1 group, pHBLV-nCoV-N-shRNA2 group and pHBLV-nCoV-N-shRNA3 group. A negative control group (a lung stem cell group transfected with only a lentiviral interference vector) and a blank control group (a normal lung stem cell group not transfected with a lentiviral interference vector) were set simultaneously.

4.8. Screening for transfection-positive ACE2 Stem cells

After the cells were cultured for 24 hours, 10% FBS-containing DMEM solution was replaced, and puromycin (2.00. mu.g.mL) was added at the optimum selection concentration^-1)，Maintaining the puromycin concentration, changing the liquid every other day until the blank control group completely dies the cells, and finishing the screening. ACE2 stem cells that were not killed by puromycin were lung stem cells that had been transfected with a recombinant lentiviral interference vector and had incorporated a novel coronavirus targeting interference sequence (nCoV-shRNA) on the DNA. The ACE2 (lung) stem cell can express dsRNA, and the dsRNA can interfere nCoV gene, thereby generating antiviral effect in the stem cell, and is called as a gene silencing gene modified stem cell.

The invention comprises selecting lentivirus vector pSilenccer 4.1.CMV. neo, carrying out enzyme digestion by EcoR I and Hind III, constructing recombinant vector LV3-nCoV-N-shRNA, co-transfecting 293T cells with the recombinant vector and packaging plasmids (pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5), packaging lentivirus, and screening positive clones by ampicillin.

4.9. Verification of shRNA expression of genetically modified Stem cells

4.9.1.RT-PCR detection of mRNA expression of shRNA

Extracting total RNAs of nCoV-N-shRNA1, nCoV-N-shRNA2 and nCoV-N-shRNA3 groups of genetically modified stem cells by using a Trizol reagent, designing primers shown in Table 2 by using GAPDH as an internal reference, and quantitatively analyzing mRNA of nCoV-N-shRNA1/2/3 under the PCR conditions that: denaturation at 95 ℃ for 3min, 12s at 95 ℃, 40s at 62 ℃ and 30s at 72 ℃ for 40 cycles. Each group is provided with 3 random complex holes. After the reaction is finished, the Ct value in each sample tube is automatically recorded by the instrument, the relative content of the target gene is calculated by taking GAPDH as an internal reference, and 2 is used^-ΔCtAnd (4) showing. And observing the mRNA expression quantity of the blank control group and the negative control group pHBLV-nCoV-N and the mRNA expression quantity of the pHBLV-nCoV-N-shRNA1, pHBLV-nCoV-N-shRNA2 and pHBLV-nCoV-N-shRNA3 groups in the 3 interference groups, deducing whether 3 specific targets have silencing effect on nCoV-N genes or not, and screening a group (pHBLV-nCoV-N-shRNA2) with the best interference effect by having statistical difference with the negative and blank control groups.

TABLE 2 design of new coronavirus S, E, M, N gene targeted interfering sequence siRNA 1-siRNA 3 amplification primers

Western Blotting detection of protein expression of shRNA

Extracting proteins of cell lysates of each group, quantifying the proteins by a BAC method, and arranging 3 random multiple holes in each group. The sample proteins were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes, primary antibody was anti-VDR diluted 1: 400, visualized using ECL chemiluminescence kit, and the relative gray values of the protein bands determined using a gel image analysis system. Observing the expression quantity of pHBLV-nCoV-N proteins of the blank control group and the negative control group and the expression quantity of the recombinant lentivirus pHBLV-nCoV-N proteins in the 3 interference groups of pHBLV-nCoV-N-shRNA1, pHBLV-nCoV-N-shRNA2 and pHBLV-nCoV-N-shRNA3, wherein the expression quantity is not statistically different from that of the positive control group and the blank control group. And the result shows that the pHBLV-nCoV-N-shRNA2 group can effectively interfere the protein expression of the nCoV-N gene.

The vectors of the present invention also include overexpression vectors such as pHBLV-CMV-IRES-ZsGreen, pHBLV-CMV-EF1-RFP, pHBLV-CMV-IRES-Puro, pHBLV-IRES-ZsGreen-PGK-Puro, pHBLV-CMVIE-ZsGreen-T2A-Puro, pHBLV-CMVIE-RFP-T2A-Puro, pHBLV-CMVIE-Luc-T2A-Puro, pHBLV-CMVIE-ZsGreen-T2A-Luc, pGC-FU, interference vectors such as pHBLV-U6-ZSGGreen, pHBLV-U6-Green-Puro, pHBLV-U6-Puro, pHV-U7-sZ A-T2-Puro, pHBLV-U6-pHV-5393-pHV-U7-SLS-T2-rV-6 5-PRV-RFP 2-LSV-RT 3-RFP 3-PRV-TMV-RT 2-prro, pHBLV-RT 3-PRV-LR-T2-5-LR-rV-LR 2-LR, pHBLV-U6-ZsGreen-T2A-Luc, pHBLV-U6-RFP-T2A-Luc, interfere with the over-expression of double-frame vectors such as pHBAd-U6-CMV (for constructing recombinant vectors for simultaneous expression of two genes, e.g., pHBAd-U6/shRNA-CMV/ACE2 for shRNA and ACE2, pHBAd-U6/shRNA-CMV/RBD for shRNA and RBD).

5. Functional detection of genetically modified stem cells

5.1. In vitro antiviral function detection of genetically modified stem cells

5.1.1. Sample collection

The throat swab of the patient with COVID-19 confirmed diagnosis is taken, double antibodies (10000IU penicillin and 10000 mug streptomycin) are added according to the proportion of 100: 1, the final concentration of the penicillin and the streptomycin is 100IU and 100 mug respectively, and the mixture is kept at 4 ℃ for overnight use.

5.1.2. Virus culture and isolation

Vero-E6 was inoculated into 12.5cm of DMEM medium containing (10% fetal bovine serum)²Placing in a culture flask at 36 deg.C and 5% CO₂Culturing to 30% confluent monolayer cells in incubator, removing culture medium, washing cells with DMEM for 2 times, adding 0.5mL of double antibody-treated COVID-19 patient sample into culture flask, standing at 36 deg.C and 5% CO₂Adsorbing for 90min in an incubator, removing the sample, adding 3.5mL DMEM culture solution (10% fetal bovine serum), observing cytopathic effect (CPE) every day, culturing for 5-7 d, taking supernatant of pathological cells, performing sucrose gradient ultracentrifugation, separating new coronavirus, and preparing 10% culture solution into 10% of the new coronavirus³～10⁵ TCID₅₀Viral fluid/ml (used simultaneously in the animal experiments described below).

5.1.3. Co-culture of genetically modified stem cells with viruses

Setting a mesenchymal stem cell group, an immortalized stem cell group, an ACE2 stem cell group and a genetically modified stem cell group (a recombinant ACE2 and shRNA stem cell group), inoculating 12 pore plates into each group, and enabling each pore to contain 2 multiplied by 10⁵Individual cells, 2mL of DMEM medium (10% fetal bovine serum), at 36 ℃ with 5% CO₂When the mixture was cultured in an incubator to 30% confluency, 0.5mL of virus solution was added to each well, and the culture was continued. Then, after incubation for 1 hour, 6 hours, 24 hours and 72 hours, 3-well supernatants were collected from each group, mixed and diluted 1: 4, 1: 12, 1: 36, 1: 108, 1: 324, 1: 972, 1: 2916 and 1: 8748 times, and subjected to RT-PCR detection.

5.1.4. Real-time fluorescent RT-PCR detection of viral RNA

Nucleic acid extraction kit (batch No. 2019004), 2019 novel coronavirus (ORF1ab/N) nucleic acid detection kit (batch No. 20200123) and DA3200 nucleic acid extractor from Daan Gen-stocky Co., Ltd, Zhongshan university, and ABI 7500 type PCR analyzer from Thermo Fisher Scientific, USA. According to the operation of the kit specification, the amplification reaction conditions are as follows: 15min at 50 ℃; 15min at 95 ℃; 15s at 94 ℃; 45s at 55 ℃; for a total of 45 cycles, fluorescence signals were collected at 55 ℃.

According to the kit specification, the result judgment criteria are as follows: if the detected sample has no amplification curve in ORF1ab and N gene channel or Ct value is greater than 38, it is judged as SARS-CoV-2 negative; if the Ct value of the detected sample in ORF1ab and N gene channel is less than or equal to 38 and there is obvious amplification curve, it is determined as SARS-CoV-2 positive; and thirdly, if the Ct value of the detected sample in ORF1ab or N gene channel is less than or equal to 38, the other channel has no amplification curve, the retest result is consistent with the original result, and the SARS-CoV-2 is judged to be positive.

5.1.5. Results of viral RNA detection

In Table 3, the maximum dilutions of the culture medium that were positive for RNA detection in 1 hour of culture of 4 groups of stem cells were all 1: 4, and were considered as the detection results of exogenously added viruses. In tables 4-5, after 4 groups of stem cells were cultured for 6 hours, the detection results of only the culture solution of the genetically modified stem cell group and intracellular RNA were still 1: 4, while the intracellular RNA content of the ACE2 stem cell group was the highest; in tables 6-8, after 4 groups of stem cells are cultured for 24-72 hours, the RNA detection result of the genetically modified stem cell group has the lowest positive titer (1: 12), and the intracellular RNA detection result of the ACE2 stem cell group has the highest positive titer (> 1: 8748), which indicates that ACE2 can promote the virus to enter the stem cells and replicate in the cells, and shRNA can interfere with the replication of the intracellular virus.

TABLE 3 detection results of viral RNA in 1 hour coculture of Stem cells with isolated New coronavirus

TABLE 4 results of viral RNA detection in culture broth from co-culture of stem cells with isolated new coronavirus for 6 hours

TABLE 5 intracellular viral RNA assay results of Stem cells cocultured with isolated New coronavirus for 6 hours

TABLE 6 detection results of viral RNA of culture broth obtained by co-culturing stem cells and isolated new coronavirus for 24 hours

TABLE 7 detection results of viral RNA of culture broth obtained by co-culturing stem cells and isolated new coronavirus for 72 hours

TABLE 8 intracellular viral RNA assay results of Stem cells cocultured with isolated New coronavirus for 72 hours

5.1.6. Co-culture characteristics of genetically modified stem cells and viruses

The stem cell group of the recombinant ACE2 becomes round, floats and dies after being cultured for 72 hours, as shown in figure 3, the expression of an ACE2 gene in the cell is favorable for absorbing virus into the cell and propagating in the cell to cause the cell to die quickly, while the stem cell group of the recombinant ACE2 and shRNA still grows adherently after being cultured for 72 hours, as shown in figure 4, the expression of shRNA can interfere the virus to replicate in the cell, so that the stem cell of the recombinant ACE2 and shRNA can absorb the virus into the cell and kill the virus in the cell.

5.2. In vivo antiviral function detection of genetically modified stem cells in animals

5.2.1. Preparation of genetically modified Stem cells

Inoculating the genetically modified stem cells into 10% FBS DMEM complete culture solution for cell culture, and replacing the culture solution for 1 time every two or three days; about 90% of cells are paved at the bottom of the bottle after about 5 days, 0.25% of pancreatin is digested for 2-3 min, 800r/min, the centrifugal radius is 12cm, the cells are centrifuged for 5min and inoculated to 75cm according to the ratio of 1: 2²In a culture flask, 5% CO at 37 ℃₂And carrying out amplification culture in a constant-temperature incubator for 4-5 d passage 1 time.

5.2.2. Grouping of laboratory animals

SPF-grade female BALB/c mice of about 40 g and 6-8 weeks old are selected and randomly divided into a genetically modified stem cell group (used for infecting NC-045512.2 strain and inoculating stem cells carrying ACE2 and shRNA), an ACE2 stem cell group (used for infecting NC-045512.2 strain and inoculating stem cells carrying ACE2), a stem cell group (used for infecting NC-045512.2 strain and inoculating stem cells not recombining ACE2 and shRNA), a positive control group (used for infecting NC-045512.2 strain and inoculating physiological saline) and a negative control group (only inoculating physiological saline).

5.2.3. Infection and inoculation of laboratory animals

The genetically modified stem cell group, the ACE2 stem cell group, the stem cell group and the positive control group are inoculated with 40 mul of NC-045512.2 strain virus solution through nasal cavity spraying respectively, and the titer is 10⁵/mlTCID₅₀The negative control group was inoculated with 40. mu.l of physiological saline by nasal spray. Then, 5% chloral hydrate solution (0.006mL/g or 0.6mg/g) is injected into the abdominal cavity of each group of mice, and the muscle is fixed on the plate after being relaxed; preparing skin of neck, wiping with iodophor, sterilizing, cutting neck skin to obtain 1cm small opening, separating tissue with forceps, and exposing air tube; will be 1 × 10⁶Injecting stem cells into mouse trachea slowly (injecting physiological saline into negative and positive control group), resetting tissue, and suturing skin; mice were observed daily for clinical symptoms and sacrificed on day 7 post infection for viral detection of lung tissue.

5.2.4. Detection of test results

RT-PCR detection

Taking 200 mul of a mouse lung tissue homogenate sample prepared by DMEM according to a Trizol method, adding 800 mul of Trizol, standing at room temperature for 5min to completely crack protein, adding 200 mul of chloroform, shaking vigorously by hand for 15sec, standing at room temperature for 3min, centrifuging at 4 ℃, 12,000g for 15min, taking the supernatant to a new Eppendorf tube, adding 0.5ml of isopropanol, standing at room temperature for 10min, centrifuging at 4 ℃, 12,000g for 10min, discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 4 ℃, 7,500g for 5min, discarding the supernatant, rinsing repeatedly for 1 time, and dissolving the precipitate treated by 30 mul of DEPC water. And (3) carrying out primer design, PCR amplification and product electrophoresis according to the kit, in order to compare different virus replication amounts of different experimental groups, simultaneously amplifying a host gene beta-actin as an internal reference, and taking the ratio of the expression level gray value of a target gene and the internal reference gene as the semi-quantitative analysis of each sample virus.

② observation of cytopathic Effect (CPE)

Thawing frozen lung tissue at-70 deg.C, homogenizing with DMEM to 10%, centrifuging at 3,000rpm for 20min, inoculating 100pl of supernatant onto 24-well culture plate with VeroE6 cell monolayer, inoculating 2 wells for each sample, adsorbing at 37 deg.C for 1 hr, sucking out sample solution, washing with PBS (2% double antibody) for 3 times, adding DMEM to 1.5ml, standing at 37 deg.C and 5% CO₂Culturing in an incubator, observing the pH of the culture solution, if the culture solution turns yellow or blue, adjusting the pH or replacing the culture solution in time, observing cytopathic effect (CPE) every day, recording whether the CPE appears, continuously observing for 7 days, and transmitting for 3-4 generations if the CPE does not appear, and observing the result. The inoculated cells generate pathological changes and are characterized in that the cells become round and fused, the light transmittance is weakened, and finally the cells die and fall off.

Indirect immunofluorescence detection

VeroE6 cells exhibiting a pathological Effect (CPE) were trypsinized and washed with PBS at 10. mu.l/well (10. mu.l)⁷/ml) is dripped on an antigen slide glass, dried and fixed in cold acetone for 10min, washed by PBS (dried and preserved at-20 ℃), anti-S protein monoclonal antibody (1: 200, prepared by PBS) is dripped on the prepared antigen slide glass, incubated in a 37 ℃ wet box for 30-40min, after three times of washing by PBS, FITC-labeled secondary antibody (1: 500, prepared by 0.02 percent Evans blue-PBS) is dripped, the temperature is 37 DEG CIncubate 20-30min in wet box, wash 3 times with PBS, seal with 50% glycerol-PBS, and observe fluorescence under fluorescence microscope. And (4) judging a result: under the condition that the negative control and the positive control are established, the VeroE6 cells inoculated by the specimen show red fluorescence to judge that the mouse is not infected by the virus; the green fluorescence in the cell membrane or cytoplasm of the VeroE6 cells inoculated with the standard sample is judged to be the infection of the mouse with the virus.

Fourthly, half infection quantity (TCID) of cell culture₅₀) Percentage of (2)

Taking 100 mu l of lung tissue homogenate supernatant of each killed mouse (10 mice in each group), diluting the lung tissue homogenate supernatant into different dilutions by a 10-fold progressive dilution method, respectively inoculating a tissue culture monolayer VeroE6 cell 96-well culture plate washed 3 times by Hank's solution, inoculating 30 mu l of cells in each well, inoculating 4 cell wells in each dilution, slightly oscillating to ensure that the homogenate is fully contacted with the cells, adsorbing for 1min at 37 ℃, washing 3 times by Hank's solution, adding cell maintenance solution, culturing in a 37 ℃ CO2 incubator, observing and recording cytopathic conditions under a common inverted microscope, continuously observing for 10-14 days, and respectively calculating the half infection amount (TCID) of VeroE6 cells in each group₅₀) Then comparing the TCIDs of the groups₅₀Percent difference, the greater the percent, the greater the viral content. From the results in tables 9-14, it can be seen that the genetically modified stem cell group has a lower infection amount of VeroE6 cell half, and the ACE2 stem cell group has a higher infection amount of VeroE6 cell half, indicating that ACE2 can absorb viruses into stem cells for replication, but can be interfered and killed by genetically modified stem cells with a targeted interference function.

TABLE 9 genetically modified Stem cell group 10 mice sacrificed lung homogenates resulting in a percentage of half the infected amount of VeroE6

TABLE 10 ACE2 Stem cell group 10 percentage of half the infected amount of VeroE6 by killing lung homogenate in mice

TABLE 11 Stem cell group 10 mice sacrificed lung homogenates resulting in a percentage of half the infected amount of VeroE6

TABLE 12 Positive control 10 mice sacrificed lung homogenates to give VeroE6 percent of half the infected amount

TABLE 13 negative control group 10 mice sacrificed lung homogenates to give VeroE6 percent of half the infected amount

TABLE 14 comparison of the percentage of VeroE6 infection in half the number of groups due to 10 mice sacrificed lung homogenates

5.3. Antiviral effect of ACE2-Ab produced by genetically modified stem cells

5.3.1. Detection principle of ACE2-Ab produced by genetically modified stem cells

The human angiotensin converting enzyme 2 antibody (ACE2-Ab) was determined using a double antigen sandwich method. Coating a microporous plate with hACE2, adding ACE2-Ab, adding HRP-labeled hACE2 to form an antigen-antibody-enzyme-labeled antigen complex, washing, developing with a substrate TMB, converting into blue, positively correlating the shade of the color with ACE2-Ab in a sample, measuring absorbance (OD) at 450nm by using an enzyme standard instrument, and calculating the concentration of the ACE2-Ab by using a standard curve.

5.3.2. Method for detecting ACE2-Ab produced by genetically modified stem cells

Standard substances of 100U/L, 50U/L, 25U/L, 12.5U/L and 6.25U/L are prepared according to the specification. Respectively arranging a blank hole, a standard hole, a sample hole to be detected (provided with 3 more holes) and a control hole (provided with 3 more holes), accurately adding 50 mu l of each standard sample into the standard hole, adding 40 mu l of sample diluent into the sample hole to be detected, adding 10 mu l of sample to be detected (lung tissue homogenate supernatant of a mouse inoculated with recombinant stem cells), adding the control sample (lung tissue homogenate supernatant of the mouse inoculated with the stem cells) into the control hole, gently shaking and uniformly mixing. The plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes. Carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry. 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells. And repeating the operation after the plate is sealed by a sealing plate film, adding 50 mu l of the color developing agent A into each hole, adding 50 mu l of the color developing agent B into each hole, slightly mixing the mixture, and developing the mixture at 37 ℃ in a dark place for 15 minutes. The reaction was stopped and the OD of each well was measured at a wavelength of 450nm with blank zeroing. And drawing a standard curve by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of the sample.

5.3.3. Detection result of ACE2-Ab produced by genetically modified stem cells

As can be seen from Table 15, the lung homogenate ACE2-Ab of mice vaccinated with the group of genetically modified stem cells (carrying the ACE2 gene) was significantly higher than the lung homogenate ACE2-Ab of mice vaccinated with the group of stem cells (not carrying the ACE2 gene). After the stem cells are inoculated with the genetically modified cells, the ACE2 gene in the stem cells can express proteins, and then mice are stimulated to produce ACE 2-Ab.

TABLE 15 comparison of ACE2-Ab assay results in mice vaccinated with the genetically modified Stem cell group and in mice vaccinated with the Stem cell group

Antiviral detection of ACE2-Ab

Firstly, ACE2-Ab (rabbit)anti-ACE 2 antibody) and ACE1-Ab (rabbit anti-ACE 1 antibody) were formulated at 0.05, 0.5, 5, 50 and 100ug/ml, respectively, and then mixed with 1X 10⁴The Vero E6 cells were mixed and washed 3 times in a 37 ℃ water bath for 1 h.

② suspending the cells by 2mL DMEM culture solution (10% FBS), transferring into 12-hole plate, adding 20uL 10 per hole³/mLTCID₅₀Virus liquid, put at 37 ℃ and 5% CO₂The incubator was 3+5 days, and cytopathic effect (CPE) was observed daily.

(iii) count the number of live cells and dead cells in 1000 cells by trypan blue staining method, and calculate the cell survival rate (survival rate ═ number of unstained cells/total number of cells observed, and viral RNA content of culture solution or cells can also be detected by RT-PCR).

The results show that when the concentrations of ACE2-Ab of the test groups are 0.05, 0.5, 5, 50 and 100ug/ml, the corresponding cell survival rates are 55%, 60%, 75%, 85% and 85% respectively; when the concentrations of ACE1-Ab in the control group are 0.05, 0.5, 5, 50 and 100ug/ml, the corresponding cell survival rates are 10-15%.

The ACE2 carried by the genetically modified stem cells can express the ACE2 protein, so that an organism is stimulated to generate the ACE2-Ab, and the ACE2-Ab has an antiviral effect.

5.4. Anti-inflammatory action of cytokines produced by genetically modified stem cells

When an organism is infected with a virus or a bacterium, various cytokines are produced, some have an anti-inflammatory effect, and some have an inflammatory effect. In the experiment, bacterial endotoxin (LPS) is used for respectively treating cells of each test group for 8 hours (table 16), and then the human interleukin in the supernatant of the cultured cells is measured by adopting a double-antibody sandwich method according to the instruction of an enzyme-linked immunosorbent assay (ELISA) kit, and the result shows that IL-1 beta and IL-8 of the immortalized stem cell group are obviously increased after the LPS treatment, but IL-10 is not obviously increased; after the recombinant ACE2 stem cell group is treated by LPS, IL-1 beta and IL-8 of the recombinant ACE2 stem cell group are not obviously increased or decreased, and IL-10 is obviously increased.

Both IL-1. beta. and IL-8 are inflammatory cytokines reported in the literature. Wherein IL-1 beta is widely involved in pathological injury processes such as human tissue destruction, edema formation and the like, and although the generation of beta defensin-4 can be promoted, the destruction is larger than the defense in general. IL-8 achieves the purposes of killing bacteria and damaging cells by chemotaxis of neutrophil and activation of the activity and phagocytosis of neutrophil lysosome enzyme, and IL-8 mainly promotes cytotoxicity, local inflammation and tumor cell proliferation.

IL-10 has a different effect than IL-1. beta. and IL-8. IL-10 is an important anti-inflammatory factor, and exerts an anti-inflammatory action mainly by inhibiting the production of inflammatory factors and chemokines by monocytes, macrophages, and the like. The results in Table 16 show that genetically modified stem cells (carrying ACE2) are able to suppress the inflammatory response by increasing IL-10 secretion under the action of LPS.

TABLE 16 anti-inflammatory cytokine assay results for genetically modified stem cells

6. Application of genetically modified stem cells

The genetically modified stem cells prepared from the remaining amniotic fluid cells, neonatal umbilical cord blood cells, umbilical cord tissues or placental tissue cells after prenatal diagnosis can be frozen in a stem cell bank at the temperature of-196 ℃ for later use according to names, ABO blood types or HLA types. When new coronavirus is epidemic, various types of DNA recombinant lung stem cell vector vaccines can be taken for culture and amplification, and then the individual treatment is carried out according to the self or homotypic use principle, so as to improve the effect of stem cell treatment or vaccination and reduce and eliminate the side effects of immune rejection and the like.

Claims

1. A genetically modified stem cell for treating COVID-19 is characterized in that a susceptible gene ACE2 of a new coronavirus is inserted into immortalized stem cell DNA transfected by SV40LT and/or hTERT to prepare the susceptible stem cell easily infected by the new coronavirus, and then an RNA interference sequence shRNA of the new coronavirus M, N, E and/or an S gene is inserted into the prepared susceptible stem cell DNA to construct the genetically modified stem cell which can absorb the new coronavirus into the stem cell through ACE2, can target and interfere the replication of the new coronavirus in the stem cell through the shRNA and can be amplified in vitro through unlimited passage.

2. The genetically modified stem cell of claim 1, wherein the stem cell comprises an embryonic stem cell, an adult stem cell, a mesenchymal stem cell, a lung stem cell, or an induced lung stem cell.

3. The genetically modified stem cell for treating COVID-19 as claimed in claim 1, wherein the inserted gene comprises construction of a recombinant lentiviral vector, cotransfection of the recombinant lentiviral vector and a packaging plasmid into 293FT cells for packaging of lentivirus, lentiviral transfection of stem cells, and screening and identification of the genetically modified stem cell.

4. The genetically modified stem cell for treating COVID-19 according to claim 1 and 3, wherein the recombinant lentiviral vector is constructed by cloning a targeting interference sequence shRNA of a new coronavirus M, N, E and/or S gene and/or a new coronavirus susceptibility gene ACE2 into a polyclonal site of the lentiviral vector.

5. The genetically modified stem cell for treating COVID-19 according to claim 1, wherein the siRNA sequence of the neocoronavirus M, N, E and/or S gene is shown in the siRNA candidate sequence of the neocoronavirus S, E, M, N gene of NC-045512.2 strain.

6. The genetically modified stem cell of claim 1, wherein the upstream primer for amplifying the hACE2 gene is: CMV-F: 5' -CGCAAATGGGCGGTAGGCGTG-3; the downstream primer is: EF 1-Rn: 5'-GCCAGTACACGACATCACTT-3', respectively; the upstream primer and the downstream primer of the beta-actin are respectively as follows: 5'-TGGACTTCGAGCAAGAGATGG-3', 5'-ATCTCCTTCTGCATCCTGTCG-3'.

7. The genetically modified stem cell of claim 1, wherein the genetically modified stem cell is prepared by the steps of:

(1) obtaining mesenchymal stem cells: and (3) separating and inducing amniotic fluid fibroblasts, mesenchymal stem cells or lung stem cells from a stem cell sample bank, remaining amniotic fluid cells after prenatal diagnosis, newborn umbilical cord blood cells, umbilical cord tissues or placenta tissue cells.

(2) Stem cells were assembled with immortalizing genes: constructing a recombinant vector carrying the hTERT and/or SV40LT, transfecting stem cells, integrating the hTERT and/or SV40LT into stem cell DNA, and preparing immortalized stem cells or lung stem cells capable of being infinitely passaged.

(3) Assembling stem cells with a new coronavirus susceptibility gene: angiotensin converting enzyme II gene is connected with lentivirus expression vector pHBLV-CMV-ACE2-EF1-ZsGreen-T2A-puro or pGC-FU, recombinant plasmid pHBLV-OE-ACE2 or pGC-FU-ACE2 is respectively constructed, recombinant plasmid pHBLV-OE-ACE2 and packaging plasmid (psPAX2 and pMD2G) or recombinant plasmid pGC-FU-ACE2 and packaging plasmid (pHelper1.0 and pHelper2.0) are respectively co-transfected into 293FT cells, recombinant lentivirus OE-ACE2 or FU-ACE2 carrying ACE2 is packaged, and the recombinant lentivirus is transfected into stem cells, so that the ACE2 gene is integrated into the DNA of the stem cells.

(4) Assembling stem cells with a novel coronavirus RNA interference gene: preferably, the targeting interference sequence siRNA of the new coronavirus (nCoV), synthesizing shRNA template, connecting to a lentiviral vector pHBLV or LV3, constructing a recombinant plasmid pHBLV-nCoV-N-shRNA or LV3-nCoV-N-shRNA, co-transfecting the recombinant plasmid pHBLV-nCoV-N-shRNA and a packaging plasmid (pHBLV, psPAX2 vector and pMD2G vector) or the recombinant plasmid LV3-nCoV-N-shRNA and a packaging plasmid (pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5) into 293FT cells, packaging the slow virus carrying the shRNA, transfecting the slow virus into stem cells, integrating shRNA gene into stem cell DNA, and enabling the stem cells to obtain new functions of interfering nCoV replication in cells.

(5) The artificially assembled immortalized gene, new coronavirus RNA interference gene and susceptibility gene are prepared into the genetically modified stem cell which has the inherent treatment function, the artificial immortalization function, the new coronavirus RNA interference function and the susceptibility characteristic of the stem cell.