Application of TMPRSS2 or TMPRSS2-containing biological material in preparation of virus proliferation promoting agent

Abstract

The invention provides application of TMPRSS2 or a TMPRSS2-containing biological material in preparation of a virus proliferation promoting agent, and belongs to the technical field of virus replication. The amino acid sequence of the TMPRSS2 is as shown in SEQ ID NO. 2. The TMPRSS2 or a TMPRSS2-containing biological material is converted into an active enzyme with strong trypsin-like activity through a self-activation mechanism, and multi-cycle replication of enveloped viruses can be promoted even in the absence of trypsin. The recombinant LLC-MK2 cell for expressing the TMPRSS2, disclosed by the invention, can stably express the TMPRSS2 after continuous passage for 20 times. The recombinant LLC-MK2 cell supports the research and culture of the TMPRSS2-dependent mediated activated membrane fusion virus, promotes the in-vitro virus reproduction, improves the separation efficiency, is convenient to operate, and provides a method for improving the virus titer, expanding the virus yield and improving the in-vitro adaptability of clinical strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus replication, and particularly relates to the application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of a virus proliferation promoter. Background Art

[0002] Since many viruses need to have their membrane proteins cleaved to be infectious, it is difficult to isolate live viruses in the laboratory, and the disease burden is likely to be underestimated. Currently, in the laboratory, exogenous proteases are mostly used to cleave membrane proteins to promote the entry of viruses into host cells and enhance the virus replication ability. However, when exogenous proteases, such as trypsin, are added in a medium containing a high concentration of fetal bovine serum, the activity of the exogenous protease is weakened. Therefore, a high concentration of trypsin needs to be added, which will cause certain damage to host cells, affect the survival and proliferation of host cells, and instead reduce the virus replication ability. Therefore, it is necessary to optimize the treatment conditions to avoid the negative impacts on host cells and viruses. Therefore, finding an alternative to exogenous proteases to promote the fusion of viruses with cell membranes and increase the virus replication efficiency is a technical problem that urgently needs to be solved currently. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide the application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of a virus proliferation promoter.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides the application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of a virus proliferation promoter, and the amino acid sequence of the TMPRSS2 is as shown in SEQ ID NO.2.

[0006] Preferably, the virus includes human parainfluenza virus type 3.

[0007] Preferably, the virus proliferation promoter is TMPRSS2 or a biological material containing TMPRSS2.

[0008] Preferably, the TMPRSS2 biological material includes a recombinant vector containing TMPRSS2 or a recombinant cell containing TMPRSS2; the nucleotide sequence of the TMPRSS2 is as shown in SEQ ID NO.1.

[0009] Preferably, the backbone plasmid of the recombinant vector is pB513B-1; the host cell of the recombinant cell is LLC-MK2.

[0010] The present invention provides a reagent for promoting virus proliferation, and the reagent includes the above-mentioned TMPRSS2 or a biological material containing TMPRSS2.

[0011] Preferably, the reagent is the above-mentioned TMPRSS2 or a biological material containing TMPRSS2.

[0012] The present invention provides a method for preparing a virus, which includes the step of culturing the above-mentioned reagent with a virus to proliferate virus cells.

[0013] Preferably, the virus includes human parainfluenza virus type 3; the reagent is a recombinant LLC-MK2 cell containing TMPRSS2.

[0014] The present invention provides a recombinant LLC-MK2 cell expressing TMPRSS2. The preparation of the recombinant LLC-MK2 cell includes: ligating TMPRSS2 shown in SEQ ID NO.1 to pB513B to obtain a recombinant vector containing TMPRSS2, and then transfecting the recombinant vector containing TMPRSS2 into LLC-MK2 cells to obtain the recombinant LLC-MK2 cell.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides an application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of a virus proliferation promoter. TMPRSS2 or the biological material containing TMPRSS2 is transformed into an active enzyme with strong trypsin-like activity through an autoactivation mechanism, and can promote the multi-cycle replication of enveloped viruses even in the absence of trypsin.

[0017] The present invention provides a recombinant LLC-MK2 cell expressing TMPRSS2. The results show that the recombinant LLC-MK2 cells can stably express TMPRSS2 after being continuously passaged 20 times. The recombinant LLC-MK2 cells support the research and culture of membrane fusion viruses activated by TMPRSS2-mediated, promote virus reproduction in vitro, improve the isolation efficiency, and are easy to operate, providing a new method for increasing virus titer, expanding virus production, and improving the in vitro adaptability of clinical virus strains. Description of the Drawings

[0018] Figure 1 It is the result of observing the green fluorescent protein expression rate of cells 48 hours after transfection under a fluorescence microscope;

[0019] Figure 2 In the left figure, the cells obtained after monoclonal selection are shown under the bright field of a fluorescence microscope, and in the right figure, the cells obtained after monoclonal selection are shown under the dark field of a fluorescence microscope;

[0020] Figure 3 It is the result of the growth curves of LLC-MK2 cells and LLC-MK2 / TMPRSS2 cells;

[0021] Figure 4Agarose gel electrophoresis results of LLC-MK2 / TMPRSS2 after 20 consecutive passages;

[0022] Figure 5 The left-middle figure is the result of the whole membrane of Western blot, and the right figure is the result of the target band cut from the whole membrane;

[0023] Figure 6 In the figure, A is the growth curve of HPIV3 ATCC strain detected by digital PCR after being cultured with LLC-MK2 cells and LLC-MK2 / TMPRSS2 cells respectively; B is the growth curve of HPIV3 ATCC strain detected by plaque assay after being cultured with LLC-MK2 cells and LLC-MK2 / TMPRSS2 cells respectively; C is the growth curve of HPIV3 clinical strain detected by digital PCR after being cultured with LLC-MK2 cells and LLC-MK2 / TMPRSS2 cells respectively; D is the growth curve of HPIV3 clinical strain detected by plaque assay after being cultured with LLC-MK2 cells and LLC-MK2 / TMPRSS2 cells respectively. Detailed implementation mode

[0024] The present invention provides an application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of a virus proliferation promoter, and the amino acid sequence of the TMPRSS2 is as shown in SEQ ID NO.2.

[0025] The research of the present invention shows that TMPRSS2 is transformed into an active enzyme with strong trypsin-like activity through an autoactivation mechanism. Therefore, TMPRSS2 can replace exogenous protease, promote the fusion of virus and cell membrane, increase the virus replication efficiency, and provide a better scheme for isolating clinical strains in the laboratory.

[0026] In the present invention, the nucleotide sequence of the TMPRSS2 is preferably as shown in SEQ ID NO.1. The biological material containing TMPRSS2 includes a recombinant vector containing TMPRSS2 or a recombinant cell containing TMPRSS2. The backbone plasmid of the recombinant vector is preferably pB513B-1; the host cell of the recombinant cell is LLC-MK2. The present invention has no special limitation on the sources of pB513B and LLC-MK2, and commercially available products disclosed in the art can be used.

[0027] In the present invention, the virus proliferation promoter is only TMPRSS2 or a biological material containing TMPRSS2. The present invention uses TMPRSS2 or a biological material containing TMPRSS2 as a protease, which can promote the multi-cycle replication of the virus. The virus preferably includes human parainfluenza virus type 3 (HPIV3), such as HPIV3 ATCC strain C243 or HPIV3 clinical strain; the HPIV3 clinical strain is from the Henan Center for Disease Control and Prevention, and the strain number is SA20190433.

[0028] The present invention provides a reagent for promoting virus proliferation, and the reagent includes the above-mentioned TMPRSS2 or a biological material containing TMPRSS2.

[0029] In the present invention, the reagent is the above-mentioned TMPRSS2 or a biological material containing TMPRSS2, and there is no need to add other components for promoting virus proliferation, such as exogenous protease, etc.

[0030] The present invention provides a method for preparing a virus, including the step of culturing the virus with the above-mentioned reagent to enable the proliferation of virus cells.

[0031] In the above preparation method, the virus includes human parainfluenza virus type 3, such as HPIV3 ATCC strain C243 or HPIV3 clinical strain. The reagent is a recombinant LLC-MK2 cell containing TMPRSS2.

[0032] The present invention provides a recombinant LLC-MK2 cell expressing TMPRSS2. The preparation of the recombinant LLC-MK2 cell includes: ligating TMPRSS2 shown in SEQ ID NO.1 to pB513B to obtain a recombinant vector containing TMPRSS2, and then transfecting the recombinant vector containing TMPRSS2 into LLC-MK2 cells, thus obtaining it.

[0033] In the present invention, ligating TMPRSS2 shown in SEQ ID NO.1 to pB513B to obtain a recombinant vector containing TMPRSS2, and the site for ligating to pB513B-1 is between the EcoRI endonuclease and the NotI endonuclease. The method for ligating TMPRSS2 to pB513B-1 in the present invention is not particularly limited, and known methods in the art can be used, or it can be commissioned to a biological company to synthesize the recombinant pB513B-1 expressing TMPRSS2.

[0034] After obtaining the recombinant vector containing TMPRSS2, transfect the recombinant vector containing TMPRSS2 into LLC-MK2 cells, thus obtaining it. The transfection method in the present invention is not particularly limited, and known methods in the art or the methods described in the transfection reagent instructions can be used, such as using Lipofectamine TMAccording to the method in the instruction manual of the 3000 transfection reagent, the recombinant vector containing TMPRSS2 was transfected into LLC-MK2 cells as a transposon and a transposase at a mass ratio of 2:1. The present invention has no special limitation on the source of the transposase, and any transposase known in the art can be used, such as the transposase described in "Yang YL, Liu J, Wang TY, et al. Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway[J]. Journal of Virology, 2021, 95(21):-. DOI: 10.1128 / JVI.00944-21.".

[0035] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0036] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] In the following embodiments, the LLC-MK2 cells were purchased from the ATCC Biological Standards Resource Center, CCRID number: 4201MON-CCTCC00156, and the full name of the LLC-MK2 cells is rhesus monkey kidney cells.

[0038] The transposase is derived from "Yang YL, Liu J, Wang TY, et al. Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway[J]. Journal of Virology, 2021, 95(21):-. DOI: 10.1128 / JVI.00944-21.".

[0039] The complete medium was prepared by adding 10 mL of fetal bovine serum, 1 mL of glutamine, and 1 mL of P.S solution to 88 mL of DMEM. Among them, glutamine was purchased from Hyclone, product number SH30034.02; the P.S solution is a penicillin and streptomycin solution, purchased from Gibco, product number 15140-122.

[0040] The preparation of the 20 μg / mL puromycin complete medium is to mix 80 mL of the above-mentioned complete medium and 20 mL of 100 μg / mL puromycin to obtain 20 μg / mL puromycin complete medium.

[0041] The fresh medium containing 2% FBS is prepared by adding 2 mL of fetal bovine serum and 1 mL of P.S. solution to 97 mL of DMEM.

[0042] The ATCC strain of HPIV3 was purchased from the American Type Culture Collection (ATCC) Biological Standards Resource Center, Strain: C 243.

[0043] Example 1

[0044] A method for constructing LLC-MK2 cells stably expressing TMPRSS2 includes the following steps:

[0045] Connect TMPRSS2 with the nucleotide sequence shown in SEQ ID NO.1 between the EcoRI endonuclease and NotI endonuclease of the pB513B-1 plasmid to obtain a plasmid containing TMPRSS2;

[0046] Using the plasmid containing TMPRSS2 or the empty vector pB513B-1 plasmid as a transposon, according to the Lipofectamine TM 3000 transfection reagent instructions, transfect into LLC-MK2 cells at a plasmid mass ratio of transposon:transposase = 2:1. Then add 20 μg / mL puromycin complete medium to screen for successfully transfected cells, and use the limiting dilution method for monoclonal selection. After observing that the cells in one well proliferate into clusters and all emit light under a fluorescence microscope, perform expansion culture to finally obtain LLC-MK2 cells stably expressing TMPRSS2, named LLC-MK2 / TMPRSS2 cells.

[0047] At the same time, LLC-MK2 cells transfected with the empty vector were also prepared, named pB513B / LLC-MK2, for standby.

[0048] The nucleotide sequence of the TMPRSS2 is:

[0049] ATGGCTTTGAACTCAGGGTCACCACCAGCTATTGGACCTTACTATGAAAACCATGGATACCAACCGGAAAACCCCTATCCCGCACAGCCCACTGTGGTCCCCACTGTCTACGAGGTGCATCCGGCTCAGTACTACCCGTCCCCCGTGCCCCAGTACGCCCCGAGGGTCCTGACGCAGGCTTCCAACCCCGTCGTCTGCACGCAGCCCAAATCCCCATCCGGGACAGTGTGCACCTCAAAGACTAAGAAAGCACTGTGCATCACCTTGACCCTGGGGACCTTCCTCGTGGGAGCTGCGCTGGCCGCTGGCCTACTCTGGAAGTTCATGGGCAGCAAGTGCTCCAACTCTGGGATAGAGTGCGACTCCTCAGGTACCTGCATCAACCCCTCTAACTGGTGTGATGGCGTGTCACACTGCCCCGGCGGGGAGGACGAGAATCGGTGTGTTCGCCTCTACGGACCAAACTTCATCCTTCAGATGTACTCATCTCAGAGGAAGTCCTGGCACCCTGTGTGCCAAGACGACTGGAACGAGAACTACGGGCGGGCGGCCTGCAGGGACATGGGCTATAAGAATAATTTTTACTCTAGCCAAGGAATAGTGGATGACAGCGGATCCACCAGCTTTATGAAACTGAACACAAGTGCCGGCAATGTCGATATCTATAAAAAACTGTACCACAGTGATGCCTGTTCTTCAAAAGCAGTGGTTTCTTTACGCTGTATAGCCTGCGGGGTCAACTTGAACTCAAGCCGCCAGAGCAGGATCGTGGGCGGTGAGAGCGCGCTCCCGGGGGCCTGGCCCTGGCAGGTCAGCCTGCACGTCCAGAACGTCCACGTGTGCGGAGGCTCCATCATCACCCCCGAGTGGATCGTGACAGCCGCCCACTGCGTGGAAAAACCTCTTAACAATCCATGGCATTGGACGGCATTTGCGGGGATTTTGAGACAATCTTTCATGTTCTATGGAGCCGGATACCAAGTAGAAAAAGTGATTTCTCATCCAAATTATGACTCCAAGACCAAGAACAATGACATTGCGCTGATGAAGCTGCAGAAGCCTCTGACTTTCAACGACCTAGTGAAACCAGTGTGTCTGCCCAACCCAGGCATGATGCTGCAGCCAGAACAGCTCTGCTGGATTTCCGGGTGGGGGGCCACCGAGGAGAAAGGGAAGACCTCAGAAGTGCTGAACGCTGCCAAGGTGCTTCTCATTGAGACACAGAGATGCAACAGCAGATATGTCTATGACAACCTGATCACACCAGCCATGATCTGTGCCGGCTTCCTGCAGGGGAACGTCGATTCTTGCCAGGGTGACAGTGGAGGGCCTCTGGTCACTTCGAAGAACAATATCTGGTGGCTGATAGGGGATACAAGCTGGGGTTCTGGCTGTGCCAAAGCTTACAGACCAGGAGTGTACGGGAATGTGATGGTATTCACGGACTGGATTTATCGACAAATGAGGGCAGACGGCTAA(SEQ ID NO.1).

[0050] The amino acid sequence of the TMPRSS2 is as follows:

[0051] MALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQPKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADG (SEQ ID NO.2).

[0052] For screening and identification, the green fluorescent protein expression rate of cells transfected for 48 h was observed with a fluorescence microscope.

[0053] Figure 1 The results showed that green fluorescence could be observed under a fluorescence microscope in the successfully transfected LLC-MK2 cells 48 h after transfection.

[0054] The cells after monoclonal selection are shown in Figure 2 .

[0055] Figure 2 The results showed that the cells under bright field of the fluorescence microscope had grown from a single monoclonal cell into a cluster, and the cells under dark field all had green fluorescence, indicating that the monoclonal cell selection was successful.

[0056] Example 2

[0057] (1) CCK8 assay to detect cell proliferation viability: After digestion and counting of the cultured LLC-MK2 cells and the LLC-MK2 / TMPRSS2 cells prepared in Example 1, they were respectively inoculated into the same 96-well plate (3 replicates were set for each), and 100 μL of cell suspension with a density of 2×10 5A cell suspension of [[ID=]], and after the culture times of 6 96-well plates were 1 - 6 days respectively, 10 μL of CCK8 was added to each well. After continuing to incubate in a cell culture incubator for 2 h, it was placed on a multifunctional microplate reader. At a wavelength of 450 nm, when calculating, the absorbance value of the sample measurement was such that the absorbance value was equal to the measured absorbance value minus the background value.

[0058] Figure 3 The results showed that transfecting TMPRSS2 into LLC-MK2 cells did not affect the growth of LLC-MK2 cells.

[0059] (2) Agarose gel electrophoresis: The LLC-MK2 / TMPRSS2 cells prepared in Example 1 were continuously passaged 20 times. DNA was extracted from the LLC-MK2 / TMPRSS2 cells at the 1st passage (P1), 5th passage (P5), 10th passage (P10), 15th passage (P15), and 20th passage (P20), LLC-MK2 cells, and the plasmid containing TMPRSS2 prepared in Example 1. Then, the DNA extracted from the LLC-MK2 / TMPRSS2 cells, LLC-MK2 cells, and the plasmid containing TMPRSS2 prepared in Example 1 at the P1, P5, P10, P15, and P20 passages was amplified respectively. The amplification products were identified by agarose gel electrophoresis and sent to the company for sequencing.

[0060] Figure 4 The results of agarose gel electrophoresis showed that the LLC-MK2 / TMPRSS2 cells were continuously passaged 20 times and stably expressed TMPRSS2.

[0061] Extract the proteins of LLC-MK2 / TMPRSS2 cells at the above different passages (P1, P5, P10, P15, P20), LLC-MK2 cells, and LLC-MK2 cells transfected with an empty vector (pB513B / LLC-MK2) for Western blotting.

[0062] Western blotting: After electrophoresis at 120 V for 60 min, the proteins were transferred to a nitrocellulose membrane. After blocking with 5% skim milk powder at room temperature for 2 h, it was incubated overnight at 4°C with a TMPRSS2 monoclonal antibody diluted 1:2000 in 5% skim milk powder. After washing 3 times with TBST, goat anti-rabbit labeled secondary antibody (1:5000) containing 5% skim milk powder was added and incubated at room temperature for 1 h. After washing 3 times with TBST, it was developed.

[0063] Figure 5The results showed that clear bands were observed both on the whole membrane and at the positions of the target bands (25 kDa and 55 kDa) cut from the whole membrane in the Western blot, indicating that LLC-MK2 / TMPRSS2 cells were stably expressing TMPRSS2 after 20 consecutive passages. Among them, the 55 kDa target band represented unactivated TMPRSS2, and the 25 kDa target band was TMPRSS2 after transformation by the self-activation mechanism.

[0064] Example 3

[0065] Virus growth curve: Under the condition of not using trypsin, it was compared whether TMPRSS2 promoted virus isolation. 100 μL of LLC-MK2 cells with a density of 2×10 5 cells / mL and the LLC-MK2 / TMPRSS2 cells prepared in Example 1 were respectively inoculated into 96-well plates and cultured until the cell density reached 90%. The HPIV3 ATCC strain and the HPIV3 clinical strain (the HPIV3 clinical strain was from the Henan Center for Disease Control and Prevention, and the strain number was SA20190433) were respectively infected with the pre-prepared cells at a multiplicity of infection = 0.01. Three replicate wells were set up. After adsorption for 2 h, the supernatant was discarded, and fresh medium containing 2% FBS was added for continued culture. At the designated time points (0 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h), the cells and the supernatant were collected for virus titer quantification. The virus titer was detected by the plaque method of immunostaining, and the virus copy number was detected by digital PCR.

[0066] Figure 6 The results showed that compared with the infection of LLC-MK2 cells with the HPIV3 ATCC strain or the HPIV3 clinical strain respectively, the reproduction efficiency was higher in the HPIV3 ATCC strain or the HPIV3 clinical strain infecting LLC-MK2 / TMPRSS2 cells; and compared with the HPIV3 ATCC strain infecting LLC-MK2 / TMPRSS2 cells, the reproduction efficiency was higher in the HPIV3 clinical strain infecting LLC-MK2 / TMPRSS2 cells; under the condition of not depending on trypsin, the HPIV3 clinical strain could not effectively reproduce in LLC-MK2 cells, and the HPIV3 replication ability in LLC-MK2 / TMPRSS2 cells was significantly better than that in LLC-MK2 cells.

[0067] Combined with Figure 5 the results showed that LLC-MK2 / TMPRSS2 cells stably expressed TMPRSS2 with strong trypsin-like activity.

[0068] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of TMPRSS2 or a biological material containing TMPRSS2 in the preparation of an agent promoting viral proliferation, characterized in that: The amino acid sequence of TMPRSS2 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The viruses include human parainfluenza virus type 3.

3. The use according to claim 1, characterized in that: The agent promoting viral proliferation is TMPRSS2 or a biological material containing TMPRSS2.

4. The use according to any one of claims 1 to 3, characterized in that: The TMPRSS2 biological material includes a recombinant vector containing TMPRSS2 or a recombinant cell containing TMPRSS2; the nucleotide sequence of the TMPRSS2 is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that: The backbone plasmid of the recombinant vector is pB513B-1; the host cell of the recombinant cell is LLC-MK2.

6. A reagent for promoting virus proliferation, characterized in that The reagent comprises the TMPRSS2 or the TMPRSS2-containing biological material as described in any one of claims 1 to 5.

7. The reagent according to claim 6, characterized in that The reagent is TMPRSS2 or a biological material containing TMPRSS2 as described in any one of claims 1 to 5.

8. A method for preparing a virus, characterized in that: The method comprises the step of culturing the virus with the reagent according to claim 6 or 7 to allow the virus cells to proliferate.

9. The preparation method according to claim 8, characterized in that: The virus includes human parainfluenza virus type 3; the reagent is recombinant LLC-MK2 cells containing TMPRSS2.

10. A recombinant LLC-MK2 cell expressing TMPRSS2, characterized in that: The preparation of the recombinant LLC-MK2 cells comprises: connecting TMPRSS2 as shown in SEQ ID NO.1 to pB513B to obtain a recombinant vector containing TMPRSS2, and then transfecting the recombinant vector containing TMPRSS2 into LLC-MK2 cells.

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