CN112546212A

CN112546212A - Novel permanent lung stem cell vector vaccine for gene silencing of coronavirus

Info

Publication number: CN112546212A
Application number: CN202011383919.5A
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 new corona virus gene silencing immortalized lung stem cell vector vaccine is characterized in that amniotic fluid fibroblast stem cells are separated from remaining amniotic fluid cells after prenatal diagnosis, an immortalized amniotic fluid stem cell line is constructed by an SV40LT and/or hTERT transfection method, an immortalized lung stem cell line is screened out, then RNA interference sequence shRNA of a new corona virus M, N, E and/or S gene is inserted into the DNA of the immortalized lung stem cell line, gene silencing immortalized lung stem cells which can interfere the replication of the new corona virus in the lung stem cells in a shRNA targeting way are prepared, and a new corona virus antibody generating gene S1-RBD is inserted into the DNA of the gene silencing immortalized lung stem cell line, so that the gene immortalized lung stem cell vector vaccine which can be industrially amplified in vitro, can interfere the replication of the new corona virus in the lung stem cell line, and can generate a new corona neutralizing antibody is prepared.

Description

Novel permanent lung stem cell vector vaccine for gene silencing of coronavirus

Technical Field

The invention relates to a new coronavirus gene silencing permanent lung stem cell vector vaccine, belonging to the infectious disease prevention and treatment technology 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 into an N-terminal S1 subunit and a C-terminal S2 subunit by host protease 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 a cell 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.

The existing novel coronavirus vaccine is mainly designed and developed by taking S protein or RBD thereof as a target. In the preparation of new coronavirus vector vaccines, human adenovirus type 5 (Ad5) is the most widely used viral vector. The human adenovirus contains 26-45kb double-stranded linear DNA genome, which contains 4 early transcription gene regions (E1-E4) and 1 late transcription gene region, and each end of the genome has 1 segment of Inverted Terminal Repeat (ITR) of about 100 bp. The E1 and E3 genes are deleted from the first generation adenovirus vector, the E1-E4 genes are deleted from the second generation adenovirus vector, and Ad5 is the third generation adenovirus vector, all virus coding genes are deleted, only 5 'end ITR and 3' end ITR and packaging signals are reserved, and 36kb exogenous genes can be accommodated. Although the cytotoxicity and immunogenicity of Ad5 are weakened, the expression time of exogenous genes is prolonged, infected cells are more and the application is wide, but the Ad5 is easy to cause nonspecific infection and is not suitable for targeted therapy, and side effects are easy to generate in clinical application. Ad5 is not integrated with host cell DNA, although it is safe, it makes target gene expression unstable, and after entering host cell, it is easy to be phagocytized by reticuloendothelial cell and loses its effect. Meanwhile, Ad5 cannot be replicated, so that the recombinant virus in vivo is fewer and fewer, and the method is not suitable for long-term treatment of clinical chronic diseases. Ad5 is essentially a virus and still has immunogenicity and cytotoxicity, the immune response of a host to a viral vector can interfere with the immune response to a target antigen, most normal people are infected by adenovirus, and the pre-existing immunity to the viral vector can also interfere with the immune effect of the vaccine.

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 immune regulation, anti-fibrosis and inflammatory factor storm inhibition. 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 literature report on the preparation of a new coronavirus vector vaccine by replacing an adenovirus vector with a further modified stem cell is found.

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 original cells, and has been 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.

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 devised the present invention based on the current therapeutic stem cells, conventional concepts and problems with conventional vaccine vectors.

The invention aims to provide a new coronavirus gene silencing immortalized lung stem cell vector vaccine prepared by replacing the existing adenovirus vector with gene silencing immortalized lung stem cells artificially assembled with immortalized genes and RNA interference genes and a preparation method thereof.

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

first, amniotic fluid fibroblasts are isolated: isolating amniotic fluid fibroblasts from amniotic fluid cells remaining after prenatal diagnosis, or isolating mesenchymal stem cells from neonatal umbilical cord blood, umbilical cord tissue or placental tissue.

Further, an immortalized amniotic fluid cell line was constructed: constructing a recombinant vector of hTERT and/or SV40LT, transfecting amniotic fibroblasts or mesenchymal stem cells, and preparing an immortalized amniotic fluid cell line or mesenchymal stem cell line capable of unlimited passage and permanent survival.

Further, screening for immortalized stem cell lines: surface molecules from different clonal cell lines were detected by flow cytometry and included the positive molecules CD73-APC, CD90-FITC, CD44-PE, CD105-Cy5.5 and the negative molecules CD11b-PE, CD19-PE, CD34-PE, CD45-PE, HLA-DR-PE.

Further, screening for immortalized lung stem cell lines: the method is characterized in that the seeds grow in a shuttle shape and are arranged into a vortex or barrier shape; positive for cell surface markers CD45, CD11a, CD14, CD90, CD34, CD71, CD25, CD105, CD117, CD166 and CD 44; ③ the keratin expression is negative, and the c-Myc, Oct4, Nanog and Nestin are positive; positive expression of vimentin, collagen III and fibrinectin, and negative expression of pro-Surfactant protein C, von Willebrand factor and alpha-smooth muscle actin.

Further, the immortalized lung stem cell line is assembled with RNA interference genes: preferably targeting interfering sequence siRNA, synthesizing shRNA template, connecting to a lentiviral vector pHBLV or LV3 to construct a recombinant plasmid pHBLV-nCoV-shRNA or LV3-nCoV-shRNA, co-transfecting the recombinant plasmid pHBLV-nCoV-shRNA and a packaging plasmid (pHBLV, psPAX2 vector and pMD2G vector) or the recombinant plasmid LV3-nCoV-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 a lung stem cell line, and integrating shRNA gene into lung stem cell DNA so as to obtain a new function capable of interfering nCoV replication in the lung stem cells.

Further, an immortalized gene and a new coronavirus RNA interference gene are artificially assembled to obtain the lung stem cell line with the stem cell treatment function, the immortalization function and the new coronavirus RNA interference function.

Furthermore, the adenovirus vector of the prior art is replaced by the DNA recombinant stem cell to prepare the vaccine: analyzing an S protein amino acid sequence (the RBD amino acid is Gly319 to Asn541) of SARS-CoV-2 according to GenBank, adopting synonymous replacement of amino acid codons to synthesize a whole gene of nucleic acid corresponding to an RBD peptide segment, respectively inserting enzyme cutting sites of EcoR and IXhoI at two ends of the RBD gene, constructing a recombinant plasmid pHBLV-RBD from the synthesized RBD gene, or respectively inserting enzyme cutting sites of HindIII and XhoI at two ends of the RBD gene, constructing a recombinant plasmid pGC-FU-RBD from the synthesized RBD gene, respectively packaging lentiviruses, transfecting a lung stem cell line with the lentiviruses, integrating the RBD gene into the DNA of the lung stem cell line, and obtaining a new corona virus gene silent immortalized lung stem cell vector vaccine capable of stimulating a host to generate corresponding antibodies by expressing the RBD protein.

The invention has the beneficial effects that:

the originally abandoned histiocyte is transformed into an immortalized lung stem cell with a plurality of new functions to replace the traditional adenovirus vector to prepare a new coronavirus gene silencing immortalized lung stem cell vector vaccine.

The lung stem cells solve the problems of ultralow temperature permanent storage, renewable utilization and commercialization requiring infinite amplification due to the assembly of immortalizing genes (SV40LT and/or hTERT).

The lung stem cell is more easily inhibited from virus replication in the stem cell due to the assembly of a new coronavirus RNA interference gene, so that the stem cell which is originally easy to kill viruses is transformed into a gene-silenced immortalized lung stem cell which is easy to kill viruses.

When the new coronavirus gene silencing immortalized lung stem cells are used as vaccine vectors, the vaccines are safer to use due to the substitution of adenovirus vectors with immunogenicity, the selection of the same type, the fact that exogenous genes do not enter host cells and the like; and because the assembled S1-RBD gene can express RBD protein which can stimulate the host to produce RBD neutralizing antibody, even if stem cells are phagocytized, the antigen information of the stem cells can be transmitted to immune cells by phagocytic cells to produce RBD neutralizing antibody, and the infection of the host cells caused by the combination of the S1-RBD and the ACE2 receptor of the host cells by the new coronavirus can be prevented.

Drawings

FIG. 1 is a clone of an immortalized lung stem cell selected from G418 and puromycin according to the present invention.

FIG. 2 is a subculture of an immortalized lung stem cell clone according to the invention transfected with SV40LT and hTERT.

FIG. 3 is a diagram showing the cell growth status of immortalized lung stem cells co-cultured with the isolated virus for 72 hours.

FIG. 4 is a graph of the cell growth status of immortalized lung stem cells with isolated virus co-cultured for 72 hours with the gene silencing of new coronavirus.

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

In FIG. 2, the immortalized lung stem cells transfected by SV40LT and hTERT grew in fusiform adherent growth during passage and grew vigorously.

In FIG. 3, the cells are seen to be in a rounded, floating, dead state, indicating that the immortalized lung stem cells die more rapidly after infection with the new coronavirus.

In FIG. 4, it can be seen that the cells still grow adherent to fusiform, which indicates that the expression of the new coronavirus shRNA can interfere the replication of the virus in the cells, and the immortalized lung stem cells with the new coronavirus gene silencing have better antiviral effect.

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 novel coronavirus gene-silenced immortalized Lung Stem cells

1. Collection of cell samples

1.1. Collection of amniotic fluid fibroblasts

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 growing adherently from the remaining amniotic fluid cells under an inverted microscope.

1.2. Collection of mesenchymal Stem cells

Aseptically taking parturient fetal lung tissue, mechanically dispersing, digesting with 0.25% trypsin, filtering with gauze with pore size of 100 μm, centrifuging at 1000 r/min for 5min, discarding supernatant, and adding DMEM culture solution (0.1umol beta-mercaptoethanol, 100UI/mL streptomycin, 10% fetal calf 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. Construction of immortalized cell line and identification of immortalized lung stem cell line

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 SV40 DNA (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 EcoR I/BamH I enzyme digestion, connection, transformation, screening and sequencing verification 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 cDNA 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, cutting enzyme and sequencing to identify, enlarging culturing hTERT-containing E.coli, and purifying pLPCX-hTERT recombinant clone with Endotoxin-Free plasmid.

2.2. hTERT/pLPCX and SV40LT/pLXSN are jointly transfected into amniotic fluid fibroblasts or lung mesenchymal stem cells

The 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 cell lines

2.3.1. Characterization of biological Properties of immortalized cell lines

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. Identification of immortalized stem cell lines

Detecting molecules on the surface of the stem cell membrane by using a flow cytometer, wherein the 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, an immortalized stem cell line which can be permanently passaged in vitro is obtained. FIG. 2 is an immortalized stem cell line that passed through generation 35.

2.3.3. Identification of immortalized pulmonary 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, CD11a, 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. Novel coronavirus RNA interference gene assembly of immortalized lung stem cell line

3.1. 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 to human genome are respectively preferred (Table 1A), and 1 irrelevant siRNA sequence is designed as a negative control (NC: 5'-TTCTCCGAACGTGTCACGTAA-3').

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

3.2. Synthesis of shRNA templates

After the target sequence is determined, shRNA templates are designed according to the polyclonal enzyme cutting sites of a lentivirus interference vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO, each template consists of two mostly complementary single-stranded DNAs with the length of 52-60nt, and each oligonucleotide is a single-stranded DNAThe 3' end of the strand DNA has 2-5U-shaped protrusions, which comprise a sense sequence of a target sequence, a loop sequence, an antisense sequence of the target sequence, a transcription termination signal and a sticky end sequence after enzyme digestion, and can form a DNA double strand with sticky ends of BamH I and ECORI enzyme digestion sites after annealing and complementation (for example, with pSilencer4.1.CMV. neo, BamH I and Hind III). 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

3.3. 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.

3.4. 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.

3.5. Packaging with shRNA lentivirus

293FT cells in logarithmic growth phase were taken at 5X 10⁶cell·mL^-1Inoculating into culture flask, adding recombinant plasmid and4 mu g of each lentivirus packaging plasmid (consisting of pHBLV, psPAX2 vector and pMD2G vector) is cotransfected with Lipofectamine200 liposome, the culture medium is replaced by complete culture medium after 8h, the culture medium is continuously cultured for 48h, and then supernatant rich in lentivirus is collected, centrifuged, filtered, subpackaged and stored at-80 ℃.

3.6. 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.

3.7. Lentiviral transfection of immortalized lung stem cell lines

At a rate of 1X 10 per hole⁶The immortalized lung stem cell line with good growth state is inoculated to a 6-pore plate according to the cell density, when the cells are fused to 30%, the cells of the test group are respectively added with 5 times 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.

3.8. Screening of transfection-positive immortalized lung stem cell lines

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) And maintaining the puromycin concentration, changing the liquid every other day until the blank control group completely dies, and finishing screening. The lung stem cell line which is not killed by puromycin is the lung stem cell line which is killed by puromycinA lung stem cell line transfected with a recombinant lentiviral interference vector and having integrated on its DNA a novel coronavirus targeting interference sequence (nCoV-shRNA). Theoretically, the lung stem cell line can express dsRNA, and the dsRNA can interfere with nCoV genes, so that the antiviral effect in the lung stem cell line is generated.

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 the recombinant vector and packaging plasmids (pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5) into 293FT cells, packaging lentivirus, and screening positive clones by ampicillin.

3.9. Verification of shRNA expression of immortalized lung stem cell line

3.9.1.RT-PCR detection of mRNA expression of shRNA

Extracting total RNA of nCoV-N-shRNA1, nCoV-N-shRNA2 and nCoV-N-shRNA3 groups of immortalized stem cells or lung stem cell lines by using 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, and deducing whether the 3 specific target points have silencing effect on the nCoV-N gene or not, wherein the three specific target points have statistical difference with the negative and blank control groups. As a result, the pHBLV-nCoV-N-shRNA2 has the best interference effect.

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

3.9.2 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. The result shows that the pHBLV-nCoV-N-shRNA2 group can effectively interfere the protein expression of the nCoV-N gene, and is called as a gene silencing permanent lung stem cell line.

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 double-frame vector such as pHBAd-U6-CMV (used to construct recombinant vectors that express both genes simultaneously, e.g., pHBAd-U6/shRNA-CMV/RBD that expresses shRNA and RBD).

4. Functional detection of gene-silenced immortalized lung stem cells

4.1. In vitro antiviral function assay

4.1.1. 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.

4.1.2. 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 coronavirus³～10⁵TCID₅₀Viral fluid/ml (used simultaneously in the animal experiments described below).

4.1.3. Co-culture of gene-silenced immortalized lung stem cells with viruses

Setting an immortalized lung stem cell group and a gene silencing immortalized lung stem cell group (carrying a target interference sequence shRNA of a new coronavirus N gene), inoculating 12 pore plates in 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.

4.1.4. 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.

4.1.5. Results of viral RNA detection

The detection results are shown in tables 3-8. In Table 3, the maximum dilutions of the culture medium that were positive for RNA detection in 1 hour of culture of 2 groups of stem cells were all 1: 12, and were considered to be the result of detection of exogenously added virus. In tables 4-8, the positive titers of the detection results of viral RNA in the culture solution of the immortalized lung stem cell group and the stem cells of the 2 groups of stem cells are rapidly increased along with the extension of the culture time, and are obviously higher than those of the immortalized lung stem cell group with the new coronavirus gene silencing, which indicates that the immortalized lung stem cell group with the new coronavirus gene silencing has better antiviral effect.

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

TABLE 4 detection results of viral RNA in culture broth from 6 hours of coculture of lung stem cells and isolated new coronavirus

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

TABLE 6 detection results of viral RNA of 24-hour coculture broth of lung stem cells and isolated new coronavirus

TABLE 7 detection results of viral RNA of culture broth for 72 hours of coculture of lung stem cells and isolated new coronavirus

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

4.1.6. Co-culture of lung stem cells and viruses

The cells of the immortalized lung stem cell group become round, float and die after 72 hours of culture as shown in figure 3, while the gene silencing immortalized lung stem cell group still grows adherently after 72 hours of culture as shown in figure 4, which indicates that the expression of shRNA can interfere the replication of virus in cells.

4.2. In vivo antiviral function assay in animals

4.2.1. Preparation of Gene-silenced immortalized Lung Stem cells

Inoculating the gene silencing immortalized lung stem cells into DMEM complete culture solution of 10% FBS 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 performing amplification culture in a constant-temperature incubator for 4-5 d passage 1 time.

4.2.2. Grouping of laboratory animals

SPF-grade female BALB/c mice of 6-8 weeks old and about 40 g are selected and randomly divided into a gene silencing immortalized lung stem cell group (used for infecting NC-045512.2 strains and inoculating gene silencing immortalized lung stem cells), an immortalized lung stem cell group (used for infecting NC-045512.2 strains and inoculating immortalized lung stem cells), and a positive control group (used for infecting NC-045512.2 strains and inoculating physiological saline) and a negative control group (only inoculating physiological saline) are arranged.

4.2.3. Infection and inoculation of laboratory animals

Gene silencing immortalized lung stem cell group and immortalized lung stemThe cell group and the positive control group were inoculated with 40. mu.l of NC-045512.2 strain virus solution by nasal spray, and the titer was 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, and separating tissue with forceps to expose trachea; 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.

4.2.4. Detection of test results

RT-PCR detection

Taking 200 mul of a mouse lung tissue homogenate specimen which is 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 treating the precipitate which is dissolved by water with 30 mul of DEPC. 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 pH of the culture solution, if it turns yellow or blue, adjusting pH in time or moreChanging the culture solution, observing cytopathic effect (CPE) every day, recording whether CPE appears, continuously observing for 7 days, and transmitting 3-4 generations if no CPE appears, 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)⁷And/ml) is dripped on an antigen slide glass, the antigen slide glass is dried and then fixed in cold acetone for 10min, washed by PBS (stored at 20 ℃ below zero after being dried), 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, washed by PBS for three times, dripped with FITC-labeled secondary antibody (prepared by 1: 500 and 0.02 percent Evans blue-PBS), incubated in a 37 ℃ wet box for 20-30min, washed by PBS for 3 times, sealed by 50 percent glycerol-PBS, and fluorescence is observed under a 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 mice are judged to be infected by the virus when green fluorescence appears in cell membranes or cell cytoplasm of VeroE6 cells inoculated by the samples.

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 by Hank's solution for 3 times, 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, respectively calculating half infection (TCID) of each group of VerE 6 cells₅₀) Then comparing the TCIDs of the groups₅₀Percent difference, the greater the percent, the greater the viral content. As can be seen from tables 9-12, the group of gene-silenced immortalized lung stem cells had a lower VeroE6 cell half infection level, indicating a better antiviral effect.

TABLE 9 Gene silencing immortalized Lung Stem cell group 10 mice sacrificed Lung tissue homogenates resulting in a percentage of half the infected amount of VeroE6

TABLE 10 immortalized Lung Stem cell group The percentage of VeroE6 infection in half the number of mice sacrificed 10 lung homogenates

TABLE 11 Positive control 10 mice sacrificed lung homogenates to give VeroE6 a percentage of half the infected amount

TABLE 12 percentage of VeroE6 infection in half the number of mice sacrificed in 10 negative controls

EXAMPLE two preparation of Gene silencing immortal Lung Stem cell vector vaccine

1. Novel coronavirus antibody-producing gene assembly of gene-silenced immortalized lung stem cells

Synthesis of RBD Gene

The amino acid sequence of the S protein of SARS-CoV-2 is analyzed by referring to GenBank accession number (MN908947.3), the RBD amino acids are Gly 319-Asn 541, and the nucleic acid corresponding to the RBD peptide segment is subjected to whole gene combination by adopting synonymous substitution of amino acid codons. Before synthesis, codon optimization is carried out, so that the content of G + C% in a genome is improved, the stability of mRNA in a mammalian cell or the rate of inputting the mRNA into cytoplasm is increased, the loss of rare tRNAs is avoided, the protein expression efficiency is enhanced, and the immunogenicity is improved. The specific method is as follows: statistical analysis of the frequency of use of NC _045512.2 strain virus codons and human codons (http:www.kazusa.or.jp/codon)the codon optimization of the S1-RBD gene of the NC045512.2 strain virus is carried out by using human preferred codons, so that the codon usage frequency is consistent with that of a human, meanwhile, the enzyme digestion recognition sequence GAATTC of EcoR I is added at the 5 'end of the gene, and the XhoI enzyme digestion recognition sequence CTCGAG is added at the 3' end. The G + C% of the optimized gene is increased from 48% to 70%, and the G + C% of the third position of the codon is increased from 39% to 100%. The optimized RBD gene sequence is 765 bp:

1.2. construction of lentivirus expression vector carrying RBD gene

The synthesized S1-RBD gene was ligated with the linearized lentiviral expression vector using T4 ligase to construct a lentiviral expression vector (pGC-FU-RBD or pHBLV-RBD), and then competent E.coli DH 5. alpha. was transformed and spread in a medium containing 50. mu.g/mL^-1And (3) performing resistance culture on an LB (Luria Bertani) plate of ampicillin, screening out positive clones, extracting plasmids, performing PCR (polymerase chain reaction), enzyme digestion or sequencing identification, performing amplification culture on the positive clones with correct sequencing, and extracting recombinant plasmids according to a plasmid extraction kit. In the same way, other nucleic acid sequences capable of stimulating the host to produce the antibody are used for constructing a lentivirus expression vector, and a corresponding vector vaccine is prepared.

1.3. Lentiviral package carrying RBD gene

293FT cells in logarithmic growth phase were taken at 5X 10⁶cell·mL^-1The density of (2) is inoculated in a culture flask. The lentiviral expression vector (pGC-FU-RBD) and the packaging plasmids (pHelper 1.0 and pHelper 2.0) were subjected to high purity endotoxin-free extraction, respectively, and 293FT cells were co-transfected according to the instructions of Upofectamine 2000, Invitmgen. At the same time, DGC-FU (containing GFP gene), packaging plasmids (pHelper 1.0 and pHelper 2.0) were co-transfected into another group of 293FT cells, and the obtained empty vector (Kntiviml. about. GFP) carrying only GFP gene was used as a control. After 8h of transfection, the medium is replaced by complete medium, the culture is continued for 48h, the supernatant rich in lentivirus is collected, centrifuged at 4 ℃ for 10min at 4000g, and filtered by a 0.45 mu m filter to obtain the lentivirus with high titerSubpackaging the virus, and storing at-80 deg.C.

1.4. Lentivirus titer detection with RBD gene

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 are transfected by the recombinant plasmid pGC-FU-RBD, the recombinant plasmid is packaged into lentivirus particles, and green fluorescence is presented 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, and then RNA was extracted according to the TRIZOL protocol of Invitrogen to determine the concentration of the extracted RNA by RT-qPCR.

1.5. Lentivirus transfection RNA interference stem cell carrying RBD gene

At a rate of 1X 10 per hole⁶The cell density is that the gene silencing immortalized lung stem cells with good growth state are inoculated to a 6-hole plate, when the cells are fused to 30%, the cells of the test group are respectively added with 5 times diluted lentivirus stock solution, and a negative control group (only transfected with lentivirus vector) and a blank control group (untransfected with lentivirus vector) are simultaneously established. After 24 hours of culture, the cells were replaced with 10% FBS DMEM solution, and ampicillin (2.50. mu.g.mL) was added at the optimum selection concentration^-1) Maintaining ampicillin concentration (ampicillin screening pGC-FU-RBD, puromycin screening pHBLV-RBD), and changing liquid every other day until blank control group cells completely die. The cells which are not killed are the gene silencing immortalized lung stem cells which are transfected by recombinant lentivirus and have integrated new coronavirus S1-RBD on DNA, namely the lung stem cell vector vaccine.

2. RBD gene detection of gene silencing immortal lung stem cell vector vaccine

RT-PCR detection of mRNA transcription of RBD genes

Taking gene-silenced immortalized lung stem cells, and dividing the cells into 1 × 10 cells⁶Inoculation at an individual cell densityAfter culturing in 6-well plates for 4 days, RT-PCR is used to detect the transcription level of RBD gene. The primer sequence of RT-PCR amplification RBD is as follows: upstream of RBD: 5'-GATTACTCATTCATTCGATATTAC-3', respectively; downstream: 5'-ATATGCAACAGATGATCGGAAC-3', respectively; beta-actin upstream: 5'-TGGACTTCGAGCAAGAGATGG-3', respectively; downstream: 5'-ATCTCCTTCTGCATCCTGTCG-3' are provided. RT-PCR detected high level transcription of RBD gene, compared with control group, showed 1 band of 409bp transcribed by RBD.

Western-Blot detection of protein expression of RBD genes

Extracting protein of the gene silencing immortalized lung stem cell vector vaccine lysate, quantifying the protein 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 detects that the protein expression of the gene silencing immortal lung stem cell vector vaccine RBD gene is obviously increased.

2.3. Immunofluorescence detection of protein expression of RBD gene

The preparation method comprises the steps of preparing a gene silencing immortal lung stem cell vector vaccine antigen tablet, taking a rabbit anti-SARS-COV-2 polyclonal specific antibody as a first antibody, taking FITC marked goat anti-rabbit IgG as a second antibody to carry out indirect Immunofluorescence (IFA) detection, and observing SARS-COV-2 specific fluorescent particles in DNA recombinant stem cells to show that an RBD gene is expressed in the cells.

3. Immune function detection of gene silencing immortal lung stem cell vector vaccine

3.1. Preparation of gene silencing permanent lung stem cell carrier vaccine

Inoculating the gene silencing immortalized lung stem cell vector vaccine into 10% FBS DMEM complete culture solution for cell culture, digesting 0.25% pancreatin when the bottom of the bottle is fully paved with about 90% of cells, and carrying out passage and amplification culture.

3.2. Preparation of laboratory animals

Selecting SPF female BALB/c mice of 6-8 weeks old and about 40 g, and randomly dividing the mice into a gene silencing immortalized lung stem cell vector vaccine group (a gene silencing vaccine group) and an immortalized lung stem cell control group (a lung stem cell control group), wherein each group comprises 10 mice.

3.3. Animal inoculation and specimen collection

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 relaxation; preparing skin of neck, wiping with iodophor, sterilizing, cutting neck skin to obtain 1cm small opening, separating muscle and connective tissue with tissue forceps, and exposing trachea; slowly injecting stem cells into mouse trachea, resetting tissue, and suturing skin; the clinical symptoms of the mice were observed daily, venous blood was collected at 1, 2, 4, 6 weeks after inoculation, centrifuged at 3,000g for 10min, and the separated serum was stored at-20 ℃ for determination of SARS-CoV-2 specific antibodies IgG and IgM.

3.4. Detection method

(ii) ELISA for IgG detection

According to the kit operation: adding 100 μ l diluted 1: 10 into each well, incubating in 37 deg.C incubator for 30min, and washing with washing solution; adding 100 μ l HRP-labeled goat anti-mouse IgG (1: 500), incubating at 37 deg.C for 30min, and washing with washing solution; adding substrate solution, incubating at 37 deg.C for 10min, adding stop solution 50 μ l, and measuring OD value at 450nm wavelength.

② ELISA detection of IgM

According to the kit operation: adding 100 μ l diluted 1: 10 into each well, incubating in 37 deg.C incubator for 30min, and washing with washing solution; add 100. mu.l of HRP-labeled goat anti-mouse IgM (1: 500), incubate at 37 ℃ for 30min, wash well with wash: adding substrate solution, incubating at 37 deg.C for 10min, adding stop solution 50 μ l, and measuring OD value at 450nm wavelength.

Measurement of neutralizing antibody

Taking 100 mu l of antibody positive serum sample, and inactivating the antibody positive serum sample in water bath at 56 ℃ for 30min to eliminate non-specific reaction factors. The test serum was filtered through a 0.22 μm filter and diluted in multiple ratios to 1: 2, 1: 4, 1: 8 … …. The virus was then diluted to 100TCID per well 1/2 inoculum₅₀(the formula for calculating the dilution factor of the virus is that the log-minus-log neutralization test of TCID50/0.2ml requires that the virus containsLog-minus log of virus/0.1 ml-log of the log between the amount of virus inoculated per well at the time of virus titration and the amount of virus inoculated per well at the time of neutralization assay, the antilog of the log being the fold by which the virus should be diluted). Adding equivalent diluted serum into the diluted virus according to 200 μ l per tube, mixing well, and performing water bath at 37 deg.C for 120 min. Duplicate wells of each sample were inoculated with VeroE6 cells, 100. mu.l of virus-serum mixture per well, except for negative cell control wells. Positive cell control: 50 μ l virus dilution +50 μ l virus +100 μ l cells. Negative cell control: add 100. mu.l of virus dilution. Cytopathic effect (CPE) was observed day by day at 37 ℃ in a 5% CO2 incubator.

3.5. The result of the detection

Specific antibody detection results: the results of IgG and IgM detection of the gene silencing immortalized lung stem cell vector vaccine at 1, 2, 4, 6 weeks after vaccination are shown in table 17. The detection results show that 16, 18 and 10 positive cases of IgG and IgM in the peripheral blood of the mice inoculated with the gene silencing immortalized lung stem cell vector vaccine and the positive cases of IgM in the peripheral blood of the mice are respectively, 4, 3 and 1 positive case of the control group are respectively, and the results show that more cases of the gene silencing vaccine group generate specific antibodies after inoculation compared with the control group.

TABLE 17 detection results of specific antibodies of gene-silenced immortalized lung stem cell vector vaccine after inoculation of mice

Detection result of neutralizing antibody: 6 mouse sera which are IgG positive in sera at 4 weeks after inoculation are operated according to the detection method of the neutralizing antibody, and VeroE6 cells which are positive in result (a control group) are all subjected to cytopathic effect (CPE) within 3-5 days of culture; while negative (control) cells grew adherent (+), and no CPE occurred; the serum of 6 cases of mice can still inhibit the attack of virus to VeroE6 cells after being diluted by 1: 16-128 times, but cannot inhibit the attack of virus to cells with the increase of the dilution times, so that the VeroE6 cells can generate CPE. The IgG in the serum samples of 6 cases of mice has the effect of neutralizing viruses, and further the gene silencing immortalized lung stem cell vector vaccine can stimulate the mice to generate neutralizing antibodies, and the titer of the neutralizing antibodies is 1: 32-128.

TABLE 18 test results of VeroE6 cell infection in half number by neutralizing antibody of DNA recombinant stem cell vector neo-corona vaccine

4. Application of new coronavirus gene silencing permanent lung stem cell vector vaccine

According to the method, the new coronavirus gene silencing immortalized lung stem cell vector vaccine prepared by the residual amniotic fluid 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 gene silencing immortalized lung stem cell vector vaccines can be selected for culture and amplification, and then the vaccines are selected according to the self or homotypic use principle for individual treatment, so that the effect of stem cell treatment or vaccination is improved, and the toxic and side effects of immune rejection and the like are reduced and eliminated.

Claims

1. A new coronavirus gene silencing permanent lung stem cell vector vaccine is characterized in that amniotic fluid fibroblast stem cells are separated from amniotic fluid cells left after prenatal diagnosis, constructing an immortalized amniotic fluid stem cell line by an SV40LT and/or hTERT gene transfection method, screening an immortalized lung stem cell line, then inserting RNA interference sequence shRNA of the new coronavirus M, N, E and/or S gene into the DNA of the immortalized lung stem cell line to prepare a gene silencing immortalized lung stem cell which can interfere the replication of the new coronavirus in the lung stem cell through shRNA targeting, and inserting a new coronavirus antibody generating gene S1-RBD into the DNA of the gene-silenced immortalized lung stem cell to prepare the gene-silenced immortalized lung stem cell vector vaccine which can be industrially amplified in vitro, can interfere the replication of the new coronavirus in a lung stem cell line and can generate a new coronavirus neutralizing antibody.

2. The new coronavirus gene silencing immortalized lung stem cell vector vaccine of claim 1, wherein the lung stem cell is selected from fetal cells, embryonic mesenchymal stem cells, adult mesenchymal stem cells remaining after prenatal diagnosis, or obtained by stem cell induction.

3. The new coronavirus gene silencing immortalized lung stem cell vector vaccine of claim 1, wherein the shRNA is inserted into the immortalized lung stem cell and comprises the steps of constructing a recombinant lentiviral vector, co-transfecting 293FT cells with the recombinant lentiviral vector and a packaging plasmid, packaging lentivirus, transfecting the lentivirus, and screening and identifying a lung stem cell line.

4. The new coronavirus gene silencing permanent lung stem cell vector vaccine of claims 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 into a multiple cloning site of the lentiviral vector.

5. The new coronavirus gene silencing immortalized lung stem cell vector vaccine of claim 1, wherein the siRNA sequence of the new coronavirus M, N, E and/or S gene is shown in "siRNA candidate sequence of NC _045512.2 new coronavirus S, E, M, N gene".

6. The new coronavirus gene silencing immortalized lung stem cell vector vaccine of claim 1, which is prepared by the following steps:

(1) isolating amniotic fluid fibroblasts: isolating amniotic fluid fibroblasts from amniotic fluid cells remaining after prenatal diagnosis, or isolating mesenchymal stem cells from neonatal umbilical cord blood, umbilical cord tissue or placental tissue.

(2) Constructing an immortalized amniotic fluid cell line: constructing a recombinant vector of hTERT and/or SV40LT, transfecting amniotic fibroblasts or mesenchymal stem cells, and preparing an immortalized amniotic fluid cell line or mesenchymal stem cell line capable of unlimited passage and permanent survival.

(3) Screening an immortalized stem cell line: surface molecules from different clonal cell lines were detected by flow cytometry and included the positive molecules CD73-APC, CD90-FITC, CD44-PE, CD105-Cy5.5 and the negative molecules CD11b-PE, CD19-PE, CD34-PE, CD45-PE, HLA-DR-PE.

(4) Screening an immortal lung stem cell line: the method is characterized in that the seeds grow in a shuttle shape and are arranged into a vortex or barrier shape; positive for cell surface markers CD45, CD11a, CD14, CD90, CD34, CD71, CD25, CD105, CD117, CD166 and CD 44; ③ the keratin expression is negative, and the c-Myc, Oct4, Nanog and Nestin are positive; positive expression of vimentin, collagen III and fibrinectin, and negative expression of pro-Surfactant protein C, von Willebrand factor and alpha-smooth muscle actin.

(5) Assembling an immortal lung stem cell line with an RNA interference gene: preferably targeting the interfering sequence siRNA, synthesizing shRNA template, connecting to a lentiviral vector pHBLV or LV3 to construct a recombinant plasmid pHBLV-nCoV-shRNA or LV3-nCoV-shRNA, co-transfecting the recombinant plasmid pHBLV-nCoV-shRNA and a packaging plasmid (pHBLV, psPAX2 vector and pMD2G vector) or the recombinant plasmid LV3-nCoV-shRNA and a packaging plasmid (pLY/helper-SL3, pLY/helper-SL4 and pLV/helper-SL5) into 293FT cells, packaging the slow virus carrying the shRNA, transfecting the slow virus into a lung stem cell line, and integrating shRNA gene into lung stem cell DNA so as to obtain a new function capable of interfering nCoV replication in the lung stem cells.

(6) The lung stem cell line with the stem cell treatment function, the immortalization function and the new coronavirus RNA interference function is obtained by preparing a manually assembled immortalization gene and a new coronavirus RNA interference gene.

(7) Preparing vaccine by replacing adenovirus vector of the prior art with DNA recombinant stem cell: analyzing an S protein amino acid sequence (the RBD amino acid is Gly319 to Asn541) of SARS-CoV-2 according to GenBank, adopting synonymous replacement of amino acid codons to synthesize a whole gene of nucleic acid corresponding to an RBD peptide segment, respectively inserting enzyme cutting sites of EcoR and IXhoI at two ends of the RBD gene, constructing a recombinant plasmid pHBLV-RBD from the synthesized RBD gene, or respectively inserting enzyme cutting sites of HindIII and XhoI at two ends of the RBD gene, constructing a recombinant plasmid pGC-FU-RBD from the synthesized RBD gene, respectively packaging lentiviruses, transfecting a lung stem cell line with the lentiviruses, integrating the RBD gene into DNA of the lung stem cell line, and obtaining the new corona virus gene silencing immortalized lung stem cell vector vaccine capable of stimulating a host to generate corresponding antibodies by expressing the RBD protein.