Broad-spectrum antiviral preparation based on bovine IFITM1 protein or bovine IFITM2 protein and application of broad-spectrum antiviral preparation

Abstract

The invention discloses a broad-spectrum antiviral preparation based on bovine IFITM1 protein or bovine IFITM2 protein and application of the broad-spectrum antiviral preparation, and belongs to the technical field of biological medicine. The invention aims to prepare a broad-spectrum effective drug for preventing or treating bovine virus diseases. The invention provides an application of a bovine IFITM1 protein or IFITM2 protein in preparation of drugs for treating or preventing bovine viruses, such as parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type I infection. The invention discloses the broad-spectrum resistance of the bovine IFITM1 protein and IFITM2 protein to various bovine viruses, overcomes the technical limitation that the traditional medicine only aims at a single virus, and provides a core technical scheme for developing a long-acting and low-drug-resistance-risk veterinary antiviral preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum antiviral preparation based on bovine IFITM1 protein or bovine IFITM2 protein and its application. Background Art

[0002] There are a wide variety of bovine viral diseases, which are complex and diverse, and are one of the main challenges faced by the global cattle industry. These diseases not only seriously affect the health of cattle, but also lead to a decline in the production performance of beef and dairy cattle, resulting in huge economic losses. With the expansion of the breeding scale and the environmental pressure brought about by climate change, the spread of viral diseases has become more common, and the control difficulty is also increasing continuously.

[0003] The following are some common bovine viral diseases:

[0004] 1. Foot-and-Mouth Disease (FMD): A highly contagious viral disease that affects cloven-hoofed animals such as cattle, pigs, and sheep. The main symptoms include fever, blisters and ulcers in the mouth and hooves. In severe cases, it will lead to a decline in the production and reproductive ability of cattle, and it is one of the key diseases for global control and epidemic prevention.

[0005] 2. Bovine Viral Diarrhea-Mucosal Disease (BVD-MD): Caused by Bovine Viral Diarrhea Virus (BVDV), it is manifested as diarrhea, loss of appetite, immunosuppression and reproductive disorders. In severe infections, it will lead to abortion and mucosal disease, bringing long-term health threats and economic losses to the cattle industry.

[0006] 3. Lumpy Skin Disease (LSD): Spread by the Lumpy Skin Disease Virus, after infecting cattle through vectors such as mosquitoes, it will form nodular lesions on the skin, accompanied by fever and swollen lymph nodes, affecting the skin health and production performance of cattle.

[0007] 4. Bovine Parainfluenza Virus Type 3 (BPIV3): BPIV3 is one of the main pathogens causing Bovine Respiratory Disease Complex (BRDC). It usually co-infects with other viruses and bacteria, resulting in symptoms such as coughing, fever, and difficulty breathing in cattle herds.

[0008] 5. Bovine Herpesvirus Type 1 (BHV-1): This virus can cause a variety of clinical symptoms, including respiratory infections, eye diseases, and reproductive system problems (such as abortion). The virus is latent and can persist in the host's nervous system for a long time after infection, and recur under stress conditions, increasing the difficulty of prevention and control.

[0009] 6. Bovine Respiratory Syncytial Virus (BRSV): It is mainly common in calves, causing severe respiratory infections, manifested as wheezing, rapid breathing, and fever, and can even be fatal in severe cases.

[0010] 7. Bovine Coronavirus (BCoV): It often causes diarrhea in calves and respiratory diseases in adult cattle, and is one of the important pathogens in cattle herds, especially spreading rapidly in the cold season.

[0011] 8. Bovine Enterovirus (BEV): It is mainly transmitted through the fecal-oral route, infecting the digestive tract system of cattle, resulting in diarrhea, vomiting, and loss of appetite, especially showing severe disease symptoms in calves.

[0012] 9. These viral diseases are not only diverse in types but also highly contagious, easily spreading on a large scale in cattle herds, causing significant economic losses. Currently, the main means of preventing and controlling these viruses include vaccination and the use of antiviral drugs. However, the existing vaccine types are limited and it is difficult to comprehensively cover all virus variants. In addition, vaccines need to be widely inoculated in cattle herds to form effective herd immunity, and for acute outbreaks of virus infections, the protective effect of vaccines often cannot be exerted in time. On the other hand, traditional antiviral drugs usually only target specific viruses and have limited effects on other virus infections.

[0013] Facing the above problems, developing a broad-spectrum and highly effective antiviral preparation has become an urgent technical need. Among the numerous viral diseases of cattle, the present invention has selected three representative viruses, namely Bovine Parainfluenza Virus Type 3 (BPIV3), Bovine Enterovirus (BEV), and Bovine Herpesvirus Type 1 (BHV-1), as the research and verification objects of the broad-spectrum antiviral preparation. These three viruses respectively represent bovine respiratory, digestive tract, and systemic multi-system infections, and have strong typicality and representativeness:

[0014] (1) Bovine parainfluenza virus type 3 (BPIV3): As one of the main pathogens of bovine respiratory disease syndrome, BPIV3 infection often causes symptoms such as dyspnea, cough, and fever in cattle herds. Existing vaccines and antiviral drugs are difficult to completely cover all virus strains, and their prevention and treatment effects are limited during acute outbreaks. The antiviral preparation of the present invention effectively reduces the severity of virus infection by inhibiting the invasion and replication of BPIV3.

[0015] (2) Bovine enterovirus (BEV): BEV infects the digestive system of cattle, often causing digestive tract symptoms such as diarrhea, vomiting, and loss of appetite, especially severe in calves, leading to dehydration and weight loss, and even death in severe cases. Currently, there is a lack of effective treatment means in the market. The antiviral preparation of the present invention shows good inhibitory effects on BEV, helping to reduce digestive tract diseases caused by the virus.

[0016] (3) Bovine herpesvirus type 1 (BHV-1): BHV-1 is a highly contagious virus that can cause infectious bovine rhinotracheitis, reproductive system diseases, and systemic symptoms. This virus has latency and can exist in the host nervous system for a long time after infection and relapse under stress conditions. The antiviral preparation of the present invention can effectively inhibit the infection and recurrence risk of BHV-1, providing a new solution for controlling the spread of this virus. It provides a new solution for solving the disease problems caused by multiple virus infections in the current cattle breeding industry, with significant economic and social benefits. Summary of the Invention

[0017] The purpose of the present invention is to prepare a vaccine or drug for an effective broad-spectrum antiviral preparation for preventing or treating cattle.

[0018] The present invention provides a bovine IFITM1 gene or a bovine IFITM2 gene. The nucleotide sequence of the bovine IFITM1 gene is as shown in SEQ ID NO.5; the nucleotide sequence of the bovine IFITM2 gene is as shown in SEQ ID NO.6.

[0019] The present invention provides the use of a bovine IFITM1 protein or a bovine IFITM2 protein in the preparation of a drug for treating or preventing infection with Bovine parainfluenza virus type 3, Bovine Enterovirus, and Bovine Herpesvirus-1. The amino acid sequence of the bovine IFITM1 protein is as shown in SEQ ID NO.12; the amino acid sequence of the bovine IFITM2 protein is as shown in SEQ ID NO.13.

[0020] Further defined, the Bovine parainfluenza virus type 3 is type A, type B, or type C.

[0021] The present invention provides an application of a bovine IFITM1 protein or a bovine IFITM2 protein in preparing a health product for assisting in the treatment or prevention of bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

[0022] The invention relates to the use of bovine IFITM1 protein or bovine IFITM2 protein in the preparation of a drug for preventing viral latent infection of bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus type 1.

[0023] The present invention provides a recombinant vector carrying bovine IFITM1 protein or bovine IFITM2 protein for use in preparing a medicine for treating or preventing bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

[0024] It is further defined that the starting vector of the recombinant vector is pET-30a.

[0025] The present invention provides an application of a recombinant microbial cell carrying a bovine IFITM1 protein or a bovine IFITM2 protein in preparing a medicine for treating or preventing bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

[0026] It is further defined that the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0027] The present invention provides a method for inhibiting the proliferation of bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type I for non-therapeutic purposes, comprising transfecting a recombinant peptide chain containing bovine IFITM1 protein or bovine IFITM2 protein into animal cells to obtain recombinant animal cells, and then utilizing the recombinant animal cells infected with bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type I to inhibit the proliferation of bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type I.

[0028] Further limitations are as follows: the animal cells are BBEC or EBTr cells; the dosage for infecting bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type I is 1 MOI; the sequence of the recombinant peptide chain containing bovine IFITM1 protein or bovine IFITM2 protein is as shown in SEQ ID NO.1 or SEQ ID NO.2; the dosage for transfection of the recombinant peptide chain is 0.01 mg·mL-1.

[0029] Beneficial effects: Appropriate concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 were transduced into BBEC and EBTr cells. qPCR and TCID50 assays consistently confirmed that BoIFITM1 / 2 could inhibit the replication of BPIV3 in a dose-dependent manner. Under conditions where low temperature restricts virus entry into cells, the effects of BoIFITM1 / 2 on the processes of BPIV3 adsorption and entry into cells were detected. In BBEC cells, BoIFITM1 / 2 affected the process of BPIV3 entry, but had no significant effect on its adsorption process; while in EBTr cells, there were significant inhibitory effects on both the adsorption and entry processes of BPIV3. When bovine IFITM was antagonized while mice were infected with BPIV3 in the lungs, the results showed that BoIFITM1 / 2 could effectively inhibit the proliferation of BPIV3 in mice.

[0030] New application scenario: The present invention first applies the broad-spectrum antiviral characteristics of IFITM1 and IFITM2 proteins to the prevention and control of bovine viruses, providing an efficient solution for the infection of highly contagious bovine viruses such as bovine parainfluenza virus type 3 (BPIV3), bovine enterovirus (BEV), and bovine herpesvirus type I (BHV-1).

[0031] Synergistic prevention and control ability for multiple virus infections: The preparation developed in the present invention can not only effectively prevent and control single virus infections, but also has the ability to deal with complex multiple infection situations, which is a key innovation point not covered by the prior art.

[0032] Optimization of the mechanism for inhibiting virus infection: By enhancing the stability and functional activity of IFITM1 and IFITM2 proteins, this preparation blocks the binding of the virus to host cells at the early stage of virus invasion and significantly reduces the virus replication rate. Compared with existing antiviral compositions, its mechanism of action is more efficient and diverse.

[0033] Improvement of technical effects: This preparation showed significant antiviral effects in experiments, and the specific data are as follows: Inhibition rate of BPIV3 infection: up to 95%, significantly higher than that of existing single antibody treatment (about 70%). Reduction in virus replication: reduced by 85% within 24 hours, more than twice that of the prior art. Prevention and control of multiple infections: In a multiple virus infection model, the prevention and control effect was improved by more than 50%.

[0034] Technical effects and expected applications: In response to the problems of strong infectivity and high treatment costs of bovine viral diseases, the present invention provides a broad-spectrum and highly efficient antiviral solution, significantly reducing economic losses.

[0035] Applicability in multiple scenarios: It can be widely applied to dairy farms, ranches, and livestock farms to help control the outbreaks of bovine virus-caused epidemics. It has important commercial value in the fields of veterinary vaccine research and development, preventive medication for cattle farms, etc. Description of the drawings

[0036] Figure 1 It is the result diagram of the induced expression and purification of TAT-BoIFITM1 and TAT-BoIFITM2; M. Page Ruler Marker; 1. Proteins of TAT-BoIFITM1 and TAT-BoIFITM2 before purification; 2. Proteins of TAT-BoIFITM1 and TAT-BoIFITM2 after purification and concentration.

[0037] Figure 2 It is the result diagram of the effects of TAT-BoIFITM1 and TAT-BoIFITM2 recombinant proteins on the activities of BBEC and EBTr cells; a: Determination of cell activity after the action of TAT-BoIFITM1 recombinant protein on BBEC cells; b: Determination of cell activity after the action of TAT-BoIFITM1 recombinant protein on EBTr cells; c: Determination of cell activity after the action of TAT-BoIFITM2 recombinant protein on BBEC cells; d: Determination of cell activity after the action of TAT-BoIFITM2 recombinant protein on EBTr cells.

[0038] Figure 3 It is the transduction result diagram of TAT-BoIFITM1 and TAT-BoIFITM2 in BBEC and EBTr cells; a: Transduction of TAT-BoIFITM1 recombinant protein in BBEC cells; b: Transduction of TAT-BoIFITM2 recombinant protein in BBEC cells; c: Transduction of TAT-BoIFITM1 recombinant protein in EBTr cells; d: Transduction of TAT-BoIFITM2 recombinant protein in EBTr cells.

[0039] Figure 4Results of the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the replication of BPIV3 in BBEC and EBTr cells; a, b: Determination of the viral loads of BPIV3 inside and outside BBEC cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; c, d: Determination of the viral loads of BPIV3 inside and outside EBTr cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; e: Determination of the virus titer of BPIV3 in BBEC cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; f: Determination of the virus titer of BPIV3 in EBTr cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2;

[0040] Figure 5 Results of the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the adsorption and invasion of BPIV3 in BBEC and EBTr cells; a: Determination of the viral adsorption load of BPIV3 in BBEC cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; b: Determination of the viral adsorption load of BPIV3 in EBTr cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; c: Determination of the viral invasion load of BPIV3 in BBEC cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2; d: Determination of the viral invasion load of BPIV3 in EBTr cells under the action of TAT-BoIFITM1 and TAT-BoIFITM2;

[0041] Figure 6 Results of the changes in the subcellular localization of TAT-BoIFITM1 and TAT-BoIFITM2 observed by laser confocal microscopy before and after virus inoculation;

[0042] Figure 7 Results of the co-localization of TAT-BoIFITM1 and TAT-BoIFITM2 with lysosomes observed by laser confocal microscopy before and after virus inoculation;

[0043] Figure 8 Results of the observation of the anatomical morphology of the lungs of infected mice; A: PBS control group; B: Group infected with BPIV3 alone; C: BPIV3 + TAT-BoIFITM1 group; D: BPIV3 + TAT-BoIFITM2 group;

[0044] Figure 9Results of pathological sections (H.E, 20×) of lung tissues of infected mice; A: PBS control group; B: BPIV3 single infection group; C: BPIV3 + TAT-BoIFITM1 group; D: BPIV3 + TAT-BoIFITM2 group;

[0045] Figure 10 Graph showing the determination results of BPIV3 loads in various tissues of infected mice; A: In the lungs; B: In the bronchoalveolar lavage fluid; C: In the heart; D: In the liver; E: In the spleen; F: In the kidneys;

[0046] Figure 11 Graph showing the determination results of the mRNA transcription levels of cytokines in the lung tissues of infected mice; A: IFN-β; B: IL-6; C: TNF-α; D: IL1-β; E: IFN-α; F: IFN-γ; G: IL-4; H: IL-10;

[0047] Figure 12 Graph showing the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the replication of BEV or BHV-1 at the cellular level; Among them, A is the graph showing the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the replication of BEV at the cellular level; B is the graph showing the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the replication of BHV-1 at the cellular level. Detailed implementation manners

[0048] Example 1. Preparation of TAT-BoIFITM1 and TAT-BoIFITM2

[0049] The corresponding protein sequences of BoIFITM1 and BoIFITM2 genes are as follows:

[0050] Protein sequence

[0051] TAT-IFITM1: (SEQ ID NO.1) YGRKKRRQRRRMIKEEHEVAVLGAPQSQAPLTTTVINIRSDTAVPDHIVWSLFNTIFLNWCCLGFVAFAY SVKSRDRKMVGDITGAQSYASTAKCLNIWALVLGIFLTIGSIVLLIFVYMAAYETALRISRHGGH;

[0052] BoIFITM1 protein sequence: (SEQ ID NO.12)

[0053] QRRRMIKEEHEVAVLGAPQSQAPLTTTVINIRSDTAVPDHIVWSLFNTIFLNWCCLGFVAFAYSVKSRDRK MVGDITGAQSYASTAKCLNIWALVLGIFLTIGSIVLLIFVYMAAYETALRISRHGGH;

[0054] TAT-IFITM2:(SEQ ID NO.2)

[0055] YGRKKRRQRRRMLKEENEVAVLGAPQSQAPVTTTVINIPRENSVPDHIVWSLFNTVFLNWCCLGFVAFAYSVKSRDRKMVGDITGAQSYASTAKCLNIWALVLGIFLTIGSIVLLIFVYMAAYETALRISRHGGH;

[0056] BoIFITM2 protein sequence: (SEQ ID NO.13)

[0057] QRRRMLKEENEVAVLGAPQSQAPVTTTVINIPRENSVPDHIVWSLFNTVFLNWCCLGFVAFAYSVKSRDR KMVGDITGAQSYASTAKCLNIWALVLGIFLTIGSIVLLIFVYMAAYETALRISRHGGH;

[0058] Nucleotide sequence:

[0059] TAT-IFITM1:(SEQ ID NO.3)

[0060] TACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTATGATTAAGGAAGAACATGAGGTGGCAGTTCT

[0061] GGGTGCTCCGCAGAGCCAGGCCCCGCTGACCACCACTGTTATAAATATTCGTTCTGATACAGCTGTTC

[0062] CTGACCACATCGTGTGGAGCCTCTTCAACACCATATTCCTGAACTGGTGCTGTTTGGGTTTTGTGGCC

[0063] TTTGCCTATAGTGTTAAATCGCGTGATAGGAAGATGGTGGGAGATATCACTGGTGCGCAGTCCTATGC

[0064] CAGTACCGCCAAATGTCTGAATATCTGGGCCCTGGTGCTGGGCATTTTTCTGACCATTGGCAGCATTG

[0065] TTCTGCTGATTTTTGTCTACATGGCTGCATATGAAACAGCATTACGTATCAGCAGACATGGAGGTCATTAA;

[0066] TAT-IFITM2: (SEQ ID NO.4)

[0067] TACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTATGTTGAAAGAAGAAAATGAAGTTGCAGTCC

[0068] TGGGTGCTCCTCAGTCCCAAGCCCCGGTTACCACCACAGTCATCAATATTCCAAGAGAAAACAGCGT

[0069] ACCGGACCACATCGTGTGGAGCCTCTTTAATACAGTTTTCCTGAACTGGTGCTGTCTGGGTTTTGTGG

[0070] CCTTTGCATATTCTGTGAAATCACGTGATCGCAAGATGGTAGGAGATATTACTGGTGCTCAGAGTTAT

[0071] GCCAGCACAGCAAAATGTCTTAACATCTGGGCCCTGGTGCTGGGCATCTTCCTGACCATCGGCAGTA

[0072] TTGTTCTTCTAATTTTTGTTTACATGGCCGCCTATGAGACCGCACTACGTATTTCTCGTCATGGGGGTCATTAA;

[0073] BoIFITM1 gene sequence: (SEQ ID NO.5)

[0074] ATGATTAAGGAAGAACATGAGGTGGCAGTTCTGGGTGCTCCGCAGAGCCAGGCCCCGCTGACCACC

[0075] ACTGTTATAAATATTCGTTCTGATACAGCTGTTCCTGACCACATCGTGTGGAGCCTCTTCAACACCATA

[0076] TTCCTGAACTGGTGCTGTTTGGGTTTTGTGGCCTTTGCCTATAGTGTTAAATCGCGTGATAGGAAGAT

[0077] GGTGGGAGATATCACTGGTGCGCAGTCCTATGCCAGTACCGCCAAATGTCTGAATATCTGGGCCCTGG

[0078] TGCTGGGCATTTTTCTGACCATTGGCAGCATTGTTCTGCTGATTTTTGTCTACATGGCTGCATATGAAACAGCATTACGTATCAGCAGACATGGAGGTCATTAA;

[0079] BoIFITM2 gene sequence: (SEQ ID NO.6)

[0080] ATGTTGAAAGAAGAAAATGAAGTTGCAGTCCTGGGTGCTCCTCAGTCCCAAGCCCCGGTTACCACC

[0081] ACAGTCATCAATATTCCAAGAGAAAACAGCGTACCGGACCACATCGTGTGGAGCCTCTTTAATACAG

[0082] TTTTCCTGAACTGGTGCTGTCTGGGTTTTGTGGCCTTTGCATATTCTGTGAAATCACGTGATCGCAAG

[0083] ATGGTAGGAGATATTACTGGTGCTCAGAGTTATGCCAGCACAGCAAAATGTCTTAACATCTGGGCCCT

[0084] GGTGCTGGGCATCTTCCTGACCATCGGCAGTATTGTTCTTCTAATTTTTGTTTACATGGCCGCCTATGAGACCGCACTACGTATTTCTCGTCATGGGGGTCATTAA;

[0085] 2. Extract the DNA of the BPIV3 genome as a template. According to the gene sequences provided in NCBI (BoIFITM1 reference sequence: NC_037356; Bo-IFITM2 reference sequence NC_037338), select BoIFITM1 / 2-F to amplify the BoIFITM1 gene sequence, and use BoIFITM1 / 2-R as the upstream and downstream primers to perform PCR amplification of the BoIFITM2 gene sequence. Gel extraction and recovery were performed on the amplified products.

[0086] BoIFITM1-F: AGAGATGATCAAGGAGGAGCACG (SEQ ID NO.7)

[0087] BoIFITM1-F: CATGGACAGCGGCTAGTGGC (SEQ ID NO.8).

[0088] 3. Construct the prokaryotic expression vectors pET-30a-TAT-BoIFITM1 and TAT-BoIFITM2

[0089] Results: Induced expression and purification of TAT-BoIFITM1 and TAT-BoIFITM2

[0090] Transform the prokaryotic expression plasmid pET-30a-TAT-BoIFITM1 and the prokaryotic expression plasmid pET-30a-TAT-BoIFITM2 into E. coli Rosetta(DE3) TM competent cells and perform induced expression. The SDS-PAGE results are as Figure 1 shown in a of, a band of the expected size of 20 kDa was detected, and both showed inclusion body expression.

[0091] Use NI 2+ -NTA affinity chromatography method to purify TAT-BoIFITM1 and TAT-BoIFITM2. Analyze and identify the purified and concentrated proteins by 12% SDS-PAGE. As Figure 1 shown in b of, TAT-BoIFITM1 and TAT-BoIFITM2 were successfully purified.

[0092] 4. Preparation and identification of rabbit anti-BoIFITM1 / 2 polyclonal antibody

[0093] Rabbits at 6 weeks of age with good mental state and normal feeding were selected for immunization. Before the first immunization, blood was collected from the rabbits and negative sera were isolated as controls. Then, BoIFITM1 and BoIFITM2 proteins were used as immunogens, and an equal volume of complete Freund's adjuvant was added and mixed with them for emulsification. The rabbits were immunized by multi-point subcutaneous injection in the back, and the immunization dose was 1 mg. After 14 days, the proteins were mixed with the same dose of incomplete Freund's adjuvant for emulsification to boost the immunity. After another 14 days, the proteins were mixed with the same dose of incomplete Freund's adjuvant for emulsification, and the rabbits were immunized for the third time. Two weeks after the third inoculation, the rabbits were bled from the heart and the sera were isolated, aliquoted and stored at -70 °C for subsequent identification to obtain BoIFITM1 antibody and BoIFITM2 antibody.

[0094] The recombinant proteins of BoIFITM1, 2, 3, 5 (pET30a-TAT-IFITM1, 2, 3, 5) were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane using a transfer instrument. The above-preserved BoIFITM1 / 2 polyclonal antibody was used as the primary antibody (1:1000), and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody (1:5000) for Western blot identification.

[0095] Results: Rabbit polyclonal antibodies were prepared using the purified and concentrated recombinant proteins of BoIFITM1 and BoIFITM2 as immunogens, and their reactivity was identified by Western blot. The cross-reactivity results showed that both the rabbit anti-BoIFITM1 and rabbit anti-BoIFITM2 polyclonal antibodies could react with the recombinant proteins of BoIFITM1, 2, 3, demonstrating their good immunogenicity, but not with BoIFITM5. Thus, it can be seen that the cross-reactivity of the BoIFITM1 and BoIFITM2 polyclonal sera with different members of the bovine IFITM family is related to the level of homology.

[0096] Example 2. Transmembrane transduction of TAT-BoIFITM1 and TAT-BoIFITM2

[0097] 1. Cytotoxicity analysis

[0098] First, induce the expression and purification of TAT-BoIFITM1 and TAT-BoIFITM2. Then, transform the recombinant prokaryotic expression plasmids pET-30a-TAT-BoIFITM1 and TAT-BoIFITM2 into E. coli Rosetta(DE3)TM competent cells for prokaryotic expression and identification of the proteins. Pick a single colony with regular shape into 5 mL of LB liquid medium (containing 30 μg / mL Kan), and culture it overnight at 37 °C with shaking at 220 r / min. Add 2 mL of the above-mentioned medium cultured overnight to 100 mL of liquid LB medium, and add 30 μg / mL Kan. Place it in a constant temperature shaker at 37 °C and culture it with shaking at 200 - 220 r / min for about 2 h. When the OD 600 nm reaches 0.6 - 0.8, add 1.0 mM IPTG for induced expression, and culture it with shaking at 220 r / min at 37 °C for 4 - 6 h. Centrifuge the bacterial solution at 2500 g in a centrifuge at 4 °C for 15 min. Discard the supernatant, wash the precipitate with PBS, and then resuspend the precipitate with an appropriate amount of PBS. Ultrasonically lyse the whole bacteria, with a power of 500 - 600 W, an ultrasonic time of 5 s, an intermittent time of 3 s, and a total duration of about 1 h for 10 mL of the bacterial solution. Centrifuge the lysate at 10000 g at 4 °C for 10 min. Aspirate the supernatant into a centrifuge tube and dissolve the precipitate with 8 M urea buffer without imidazole.

[0099] After treating the sample, perform SDS-PAGE, analyze its expression form after Coomassie brilliant blue staining, and then perform Western blot identification with a His-tag monoclonal antibody as the primary antibody (1:5000) and a labeled goat anti-mouse IgG as the secondary antibody (1:5000) to determine whether the target protein is expressed.

[0100] Use NI 2+ -NTA affinity chromatography method to purify the induced-expressed TAT-BoIFITM1 and TAT-BoIFITM2. Load the purified protein into a boiled dialysis bag, and perform gradient dialysis with TGE solution in a chromatography cabinet at 4 °C, changing the solution every 4 h, and minimizing the concentration gradient as much as possible to completely remove urea and imidazole. Completely cover the dialyzed protein with sucrose, concentrate it in a chromatography cabinet at 4 °C, and then measure the concentration of the protein using a BCA protein concentration assay kit. Aliquot it and store it at -70 °C.

[0101] Dilute TAT-BoIFITM1 and TAT-BoIFITM2 to 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 mg / mL respectively using serum-free DMEM medium. Seed BBEC and EBTr cells into a 96-well cell plate. When the density reaches 80%, discard the old medium, and add 100 μL of TAT-BoIFITM1 and TAT-BoIFITM2 at the above concentrations to each well. Set up a column of empty cell controls without adding any substances. After placing them in a 37 °C, 5% CO2 cell incubator for 10 min, 20 min, 30 min, and 1 h respectively, discard the protein dilution solution, add 100 μL of serum-free DMEM culture medium to each well, and add 10 μL of CCK-8 reagent to each cell well under as light-proof conditions as possible. Place it in a 37 °C incubator for light-proof incubation for 2 h. Use a spectrophotometer to read the OD 450 . Record the data, analyze the data, compare the relative viability of the cells treated with TAT-BoIFITM1 and TAT-BoIFITM2 with that of the control group cells, and plot the cytotoxicity data graph.

[0102] Results: In this experiment, the CCK-8 method was used to determine the non-cytotoxic concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 recombinant proteins at 0.005, 0.01, 0.02, 0.05, 0.10, 0.20, 0.30 mg / mL in BBEC and EBTr cells. The CCK-8 assay results are as Figure 2 shown. When the concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 recombinant proteins are within 0.02 mg / mL, there is no obvious change compared with the cell viability of the control group, and the results of the viability assays of BBEC and EBTr cells tend to be consistent. Therefore, TAT-BoIFITM1 and TAT-BoIFITM2 recombinant proteins within 0.02 mg / mL are finally selected for subsequent experiments in BBEC and EBTr cells.

[0103] 2. Exploration of the transduction concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 recombinant proteins

[0104] BBEC and EBTr cells were seeded in 12-well plates and placed in an incubator at 37°C with 5% CO2. After the cell density reached 80%, TAT-BoIFITM1 and TAT-BoIFITM2 were diluted with serum-free DMEM, and the concentrations were set at 0.005 mg / mL, 0.01 mg / mL, and 0.02 mg / mL, respectively. Then they were added to the 12-well plates. After culturing for 20 min, the cells were collected. Rabbit anti-BoIFITM1 and BoIFITM2 were used as the primary antibodies (1:2000), and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody (1:5000). Western blot was used to identify the transduction function of the proteins.

[0105] Results: TAT-BoIFITM1 and TAT-BoIFITM2 were transduced into BBEC and EBTr cells at concentrations of 0.005, 0.01, and 0.02 mg / mL, respectively. After incubation for 20 min, the cells were collected for Western blot detection. In this experiment, TAT-mScarlet recombinant fluorescent protein was used as a control. As Figure 3 shown, the target bands of TAT-BoIFITM1 and TAT-BoIFITM2 could be detected at concentrations of 0.005, 0.01, and 0.02 mg / mL, and showed a dose-dependent manner. The amount of transduction into cells increased with the increase in the concentration of the recombinant protein.

[0106] Preparation of TAT-mScarlet:

[0107] Amplification and recovery of the target gene

[0108] Using pCMV-mScarlet as a template, the upstream primer: ATCGGGATCCTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGTTAGCAAAGGTGAAGCTGTCA (SEQ ID NO.9); the downstream primer: ATCGCTCGAGTTATTTATACAGCTCATCCATGCCACC (SEQ ID NO.10) were used for PCR to amplify TAT-mScarlet. The PCR product was recovered by gel extraction using a DNA purification and recovery kit.

[0109] TAT-mScarlet: (SEQ ID NO.11)

[0110] TACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGTTAGCAAAGGTGAAGCTGTCATCAAAGAGTTCA

[0111] TGCGCTTTAAAGTTCACATGGAAGGCAGCATGAATGGACACGAATTTGAAATTGAAGGCGAAGGTG

[0112] AAGGTCGTCCCTATGAGGGTACTCAGACTGCAAAACTGAAAGTAACCAAGGGAGGTCCTCTGCCGT

[0113] TTTCTTGGGACATCCTCAGCCCCCAGTTCATGTATGGCAGCCGAGCCTTCACCAAGCATCCGGCAGA

[0114] CATTCCTGATTACTACAAACAGAGCTTTCCAGAAGGCTTCAAATGGGAGCGTGTGATGAACTTTGAG

[0115] GATGGTGGTGCAGTTACAGTTACCCAAGACACCTCGCTGGAAGATGGTACCCTCATCTATAAAGTGA

[0116] AGCTGCGTGGCACCAACTTCCCGCCGGATGGCCCGGTCATGCAGAAGAAGACCATGGGCTGGGAAG

[0117] CAAGCACAGAGCGTCTCTATCCTGAGGATGGGGTTCTGAAGGGAGATATCAAGATGGCTCTGCGCCT

[0118] GAAAGATGGTGGTCGTTACCTGGCAGACTTTAAAACCACCTACAAAGCCAAGAAACCGGTTCAGAT

[0119] GCCTGGAGCCTATAATGTAGATAGAAAACTGGATATCACCAGCCATAATGAAGACTATACTGTGGTGGAACAATATGAAAGATCAGAAGGACGCCATAGCACAGGTGGCATGGATGAGCTGTATAAA。

[0120] 2. Double digestion and ligation of the target gene and the vector

[0121] According to the corresponding restriction endonucleases BamH I and Xho I of the plasmid, the recovered amplified products and the vector were respectively identified by double digestion (37 °C for 2 h). The double digestion identification system is as follows (50 μL).

[0122] Use a DNA purification and recovery kit for the recovered products. The digested products and the vector were mixed and ligated overnight in a metal bath at 16 °C.

[0123] Transformation: All 10 μL of the ligation product was transferred into the competent cell E. coli DH5α, ice-bathed for 30 min, heat-shocked at 42 °C for 90 s, ice-bathed for 5 min, 200 μL of sterile LB liquid culture medium was added to the competent cells, cultured at 37 °C with shaking at 220 r / min for 1 h, and then the culture product was spread on an LB agar plate containing 30 μg / mL kanamycin (Kan) and cultured overnight at 37 °C.

[0124] 3. Identification of recombinant plasmids

[0125] Single colonies of the transformed pET-30a-TAT-mScarlet, pET-30a-CTP-mScarlet, and pET-30a-mScarlet were picked into an LB medium containing 30 μg / mL Kan, cultured at 37 °C with shaking at 220 r / min for 12 h, and then the recombinant plasmid pET-30a-TAT-mScarlet was extracted and identified by double digestion using BamH I and Xho I. At the same time, PCR identification was carried out. The PCR system and procedure were the same as above. After successful identification, the recombinant plasmid was sent to BGI Tech Solutions (Beijing) Co., Ltd. for sequencing.

[0126] Example 3. Effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the replication of BPIV3 at the cellular level

[0127] 1. Detection of the virus titer of BPIV3 after treatment with TAT-BoIFITM1 and TAT-BoIFITM2 using the TCID50 method

[0128] BBEC and EBTr cells were seeded in a 12-well cell plate. After the cell density reached approximately 80%, 0.005 mg / mL, 0.01 mg / mL, and 0.02 mg / mL of TAT-BoIFITM1 and TAT-BoIFITM2 were transduced into the cell plate. At the same time, the TAT-mScarlet protein was used as a control. After 20 min, the cells were infected with BPIV3 at 1 MOI. After 24 h, the samples were collected, and the virus titers were measured in MDBK cells and EBTr cells respectively. The titer of BPIV3 was calculated according to the Reed-Muench method.

[0129] 2. qPCR Detection of BPIV3 Load after the Action of TAT-BoIFITM1 and TAT-BoIFITM2

[0130] Transduce TAT-BoIFITM1 and TAT-BoIFITM2 with concentrations of 0.005 mg / mL, 0.01 mg / mL, and 0.02 mg / mL into a 24-well cell culture plate. After 20 min, add 1 MOI of BPIV3. The steps are the same as above. Collect cell samples at 6 h, 12 h, 24 h, 36 h, and 48 h respectively, and use qPCR to measure the viral load of BPIV3.

[0131] Wash the cell samples 3 times with PBS solution. Add 500 μL of Trizol reagent to each well and let it stand at room temperature for 10 min, then collect the cell samples into RNase-free EP tubes. Add 100 μL of chloroform to each tube and shake vigorously, let it stand at room temperature for 15 min, and centrifuge at 12,000 r / min for 15 min using a 4 °C centrifuge. At this time, the solution is divided into three layers. Aspirate the upper layer solution into a new EP tube, add an equal volume of isopropanol, and leave it overnight in a -20 °C refrigerator. Centrifuge at 12,000 r / min for 15 min in a 4 °C centrifuge, discard the supernatant, wash the precipitate with the pre-prepared 75% ethanol solution, and centrifuge at 7500 r / min for 5 min. Discard the supernatant and air-dry the ethanol as much as possible, and dissolve the precipitate with RNase-free ddH2O. Refer to the reverse transcription kit Plus All-in-one 1st Strand cDNA Synthesis SuperMix to reverse transcribe RNA into cDNA.

[0132] Reaction program: This experiment adopts a two-step method. Pre-denature at 95 °C for 30 s; denature at 95 °C for 5 s, anneal at 60 °C for 34 s, and repeat 40 cycles. After the reaction program ends, obtain the respective gene copy numbers and the corresponding internal reference gene (GAPDH) copy numbers in the test samples. The viral load is calculated using the 2 -△Ct method. If the calculated result is small, the value can be expanded 1000 times for subsequent analysis and comparison. Use GraphPad Prism software version 7.0 to perform statistical comparison analysis and draw statistical graphs for each group of data. The significant difference analysis uses the paired two-way ANOVA method for testing, with p < 0.05, which is statistically significant.

[0133] Results: After transduction of 0.01 mg / mL of TAT-BoIFITM1 and TAT-BoIFITM2 into BBEC and EBTr cells respectively, the cells were infected with BPIV3 at 1 MOI. At 6, 12, 24, 36, and 48 h post-infection, the RNA of cells and supernatants in each group was collected to detect the BPIV3 load. The qPCR results showed that after transduction of TAT-BoIFITM1 and TAT-BoIFITM2, the BPIV3 loads inside and outside BBEC and EBTr cells decreased significantly, especially at 24 h and 36 h, and the inhibitory effect of TAT-BoIFITM2 was stronger than that of TAT-BoIFITM1, as Figure 4 shown.

[0134] After transduction of different concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 into BBEC and EBTr cells respectively, the cells were infected with BPIV3 at 1 MOI. After 24 hours of treatment, the samples were collected, and the virus titers were measured in MDBK and EBTr cells respectively. The Reed-Muench method was used to calculate the titers of BPIV3. The results were as Figure 4 shown in e. Compared with the control group, as the transduction concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 increased, the virus titers showed a downward trend, and the same results were presented in BBEC and EBTr cells, demonstrating that TAT-BoIFITM1 and TAT-BoIFITM2 inhibited the replication of BPIV3 in a dose-dependent manner in BBEC and EBTr cells.

[0135] Example 4. Effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the adsorption and invasion of BPIV3

[0136] TAT-BoIFITM1 and TAT-BoIFITM2 at concentrations of 0.005 mg / mL, 0.01 mg / mL, and 0.02 mg / mL were transduced into BBEC and EBTr cells respectively. 1 MOI of BPIV3 was added to each well, and a separate infection control group was set up. After incubating the cell plates at 4°C for 1 h, the cells were washed 3 times with PBS, cell samples were collected with Trizol, and cell RNA samples were collected. The viral mRNA levels were detected by qPCR, and the data were collected for statistical analysis.

[0137] The experimental procedure for the entry of TAT-BoIFITM1 and TAT-BoIFITM2 into cells by BPIV3 was the same as the adsorption process in the early stage. After incubation at 4°C for 1 h, the cells were washed once with cold PBS, then replaced with medium at 37°C and cultured in a cell culture incubator at 37°C and 5% CO2 for 2 h. After incubation, the cells were washed three times with trypsin and proteinase K to wash away the virus that had not entered the cells. Cell RNA samples were collected with Trizol, and the viral mRNA levels were detected by qPCR. The data were collected for statistical analysis.

[0138] Results: In this experiment, by the method of restricting virus entry into cells at low temperature, the effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the adsorption and entry processes of BPIV3 in BBEC and EBTr cells were determined. As Figure 5 shown, for the adsorption process, after transduction of different concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 into BBEC cells, there was no significant difference in the viral load of BPIV3 compared with the control group, but the viral load showed a downward trend in EBTr cells. During the process of virus entry into cells, after transduction of different concentrations of TAT-BoIFITM1 and TAT-BoIFITM2 into BBEC and EBTr cells, the viral load of BPIV3 showed a downward trend. Therefore, in BBEC cells, TAT-BoIFITM1 and TAT-BoIFITM2 did not show a significant inhibitory effect on the adsorption of BPIV3, while in EBTr cells, an obvious inhibitory effect was shown.

[0139] Example 5. Subcellular localization of BoIFITM1 and BoIFITM2 and the effect of BPIV3 on their localization

[0140] Before infection of BBEC with BPIV3, TAT-BoIFITM1 and TAT-BoIFITM2 were mainly distributed in dots on the cell membrane. However, after BPIV3 infection of the cells, after BPIV3 infection, both TAT-BoIFITM1 and TAT-BoIFITM2 originally located on the cell membrane were transferred to the cytoplasm. The localization of the TAT-mScarlet control protein did not change significantly before and after virus infection, as Figure 6 shown.

[0141] Subsequently, the co-localization of TAT-BoIFITM1 and TAT-BoIFITM2 with lysosomes before and after BPIV3 infection of cells was observed by laser confocal microscopy. The results were as Figure 7 shown. After BPIV3 infection of cells, although TAT-BoIFITM1 and TAT-BoIFITM2 were transferred from the cell membrane to the cytoplasm, no obvious co-localization phenomenon with lysosomes was observed.

[0142] Example 6. Effects of TAT-BoIFITM1 and TAT-BoIFITM2 on the proliferation of BPIV3 in mice

[0143] 1. Mouse lung infection test using a laryngoscope

[0144] In this study, a visual laryngoscope was used for the test. First, each group of mice was intraperitoneally injected with 5% chloral hydrate prepared in advance in batches at a dose of 0.3 mL / 100 g to achieve anesthesia. After a few minutes, when the mice entered a coma state, they were fixed on a fixing table. And in order to better fix the mice and make the operation more convenient, the teeth of the mice were fixed with a thin string, both hands held the limbs and tails of the mice, and the fixing table was tilted to make it an inclined plane of about 60°. After sucking the sample with a micro-injection needle, it was inserted into the indwelling needle. Open the mouse's mouth, gently pull the mouse's tongue outward to one side with forceps, insert the leaf-shaped tip of the visual laryngoscope into the oral cavity and throat, and after seeing the larynx, slowly insert the indwelling needle into the larynx, paying attention to getting as close to the larynx as possible without touching it. Slowly drip the sample into the mouse's lungs as much as possible to prevent the mouse from choking to death due to too fast a speed. After the injection, place the mouse on its side in a warm mouse cage, and pay attention to observing whether the mouse recovers after half an hour.

[0145] 2. Mouse lung infection dose and procedure

[0146] 1) Animal grouping, immunization and challenge

[0147] C57 BL / 6 mice aged 6 - 8 weeks were divided into four groups: blank control group, BPIV3 group, BPIV3 + TAT-BoIFITM1 group, and BPIV3 + TAT-BoIFITM2 group.

[0148] Blank control group: 3 mice in each group at 12, 24, 36, 48, and 72 h after injecting 20 μL of sterile PBS;

[0149] BPIV3 group infected alone: 5 mice in each group at 12, 24, 36, 48, and 72 h after being infected with 20 μL of BPIV3 (titer 10 8 TCID 50 / 0.1 mL);

[0150] BPIV3 + TAT-BoIFITM1 group: 5 mice in each group at 12, 24, 36, 48, and 72 h after intratracheally injecting 20 μg of TAT-IFITM1 protein and 20 μL of BPIV3;

[0151] BPIV3 + TAT - BoIFITM2 group: After tracheal injection of 20 μg of TAT - IFITM2 protein and 20 μL of BPIV3, 5 mice in each group were sacrificed at 12, 24, 36, 48, and 72 h.

[0152] 2) Observe and record the clinical changes of mice in each group, such as whether the mice have loss of appetite, gradual weight loss, etc. Weigh the mice before and after virus challenge and record the weight changes.

[0153] Results: Observe the lung tissues of mice in each group. As Figure 8 shown, the lung tissue morphology of the PBS control group was normal without any lesions. In the group infected with BPIV3 alone, mild swelling of the lungs was observed, showing dark red color, and obvious bleeding points, which were most significant at 24 h. Compared with the group infected with BPIV3 alone, the symptoms in the BPIV3 + TAT - BoIFITM1 and BPIV3 + TAT - BoIFITM2 groups were significantly alleviated, the lungs were light pink, and there were no obvious bleeding points. This result indicates that BoIFITM1 and BoIFITM2 significantly inhibited the damaging effect of BPIV3 on mouse lung tissues.

[0154] 3. Histopathological observation of mouse lung tissues

[0155] Dissect the mice immediately after decapitation, take out the lungs, cut the lungs into pieces the size of beans and place them in EP tubes containing 4% paraformaldehyde fixative, and store them in a 4℃ refrigerator or at room temperature. The subsequent production and staining of tissue sections were completed in cooperation with Wuhan Sevier Biotechnology Co., Ltd.

[0156] 1) Preparation of paraffin - embedded sections of lung tissues

[0157] 2) Hematoxylin - eosin (H.E.) staining of tissue sections

[0158] Deparaffinization of paraffin sections: Place the paraffin sections in an oven at 60℃ for 1 - 2 h; Deparaffinize the paraffin sections with conventional xylene and ethanol to water; Stain the cell nuclei with hematoxylin: Stain with hematoxylin for 10 min, rinse with running water, remove the excess stain, differentiate with 0.7% hydrochloric acid ethanol for several seconds, rinse with running water, and let the sections turn blue for about 15 min, soak in 95% ethanol for 30 s; Stain the cytoplasm with eosin: Stain with alcoholic eosin for 30 s; Dehydrate and mount the slides: I 95% ethanol for 30 s, II 95% ethanol for 30 s, I 100% ethanol for 30 s, II 100% ethanol for 30 s, soak in phenol xylene for 30 s, I xylene for 30 s, II xylene for 30 s, and mount with neutral gum. Observe under a light microscope that the cell nuclei are blue and the cytoplasm is generally red.

[0159] Results: Perform H.E. staining on the lung tissue samples of mice in each group. The results are as Figure 9As shown, the alveolar structure in the PBS control group was intact without any inflammatory lesions. Obvious pathological changes were observed in the group infected with BPIV3 alone, including thickening of the alveolar septum, a large number of inflammatory cell infiltrations, and severe congestion, indicating that BPIV3 infection caused serious damage to the lung tissue of mice. In contrast, the overall lesions in the BPIV3+TAT-BoIFITM1 and BPIV3+TAT-BoIFITM2 groups were mild. Although there were a small number of inflammatory cell infiltrations, the severity was much less than that in the group infected with BPIV3 alone. Therefore, it can be seen from the pathological section results that both BoIFITM1 and BoIFITM2 can inhibit the damage caused by BPIV3 to the lung tissue of mice.

[0160] 4. qPCR detection of the viral load of BPIV3 in various tissues of mice

[0161] After decapitating the mice, disinfect their chest and abdomen, cut open their abdominal cavity, thoracic cavity and neck skin with scissors, dissect out the trachea of the mice, and clamp a thin string through the bottom of the trachea with forceps. After sucking 0.5 mL of sterile PBS with a disposable 1 mL syringe, carefully insert it into the trachea from a position close to the head, and tie the inserted part with a thin string. Slowly inject the PBS into the lungs of the mice, gently knead the lungs of the mice for 10 s, and aspirate the syringe. After repeating 3 times, collect the alveolar lavage fluid into an EP tube. Then, cut out the heart, liver, spleen, lung and kidney of the mice respectively, take pictures and weigh them for subsequent experiments.

[0162] Take out the tissues of the dissected mice, weigh 0.5 g each, add liquid nitrogen to quickly freeze the tissues, grind them with a mortar, and add 1 mL of Trizol after grinding into powder for extracting the RNA of the tissues; place the alveolar lavage fluid in a 4°C centrifuge, centrifuge at 3000 r / min for 15 min, and add 1 mL of Trizol for extracting RNA. The steps of RNA extraction and reverse transcription are the same as in 2.2.4.3. During the process of extracting the RNA of the tissues, repeat the operation twice when extracting proteins with chloroform.

[0163] Use the qPCR method to detect the BPIV3 RNA in the heart, liver, spleen, lung, kidney and alveolar lavage fluid of mice. Using BPIV3-qF and BPIV3-qR as detection primers and murine β-actin as an internal reference gene, compare the viral loads of BPIV3 in the heart, liver, spleen, lung, kidney and alveolar lavage fluid of each group of mice.

[0164] Results: The BPIV3 loads in the hearts, livers, spleens, lungs, kidneys and bronchoalveolar lavage fluids of mice in each group were measured by qPCR. The results showed that BPIV3 could infect all the above tissues, and with the extension of time, it showed a trend of first increasing and then decreasing. The infection levels in the heart, spleen and lung were relatively high at 24 h and 36 h, and relatively high viral loads could still be detected in the liver, kidney and bronchoalveolar lavage fluid at 48 h. However, compared with the group infected with BPIV3 alone, the viral loads in BPIV3+TAT-BoIFITM1 and BPIV3+TAT-BoIFITM2 groups at 12 - 48 h were significantly decreased, as Figure 10 shown. It indicated that TAT-BoIFITM1 and TAT-BoIFITM2 effectively inhibited the proliferation of BPIV3 in mice.

[0165] 5. Determination of cytokines in mouse lung tissues

[0166] After dissecting the mice, 0.5 g of each tissue was taken out, added with liquid nitrogen for quick freezing of the tissue, and then ground in a mortar. After grinding into powder, 1 mL of Trizol was added for extracting the RNA of the tissue.

[0167] The RNA of various cytokines in the mouse lung was detected by qPCR method, using mouse β-actin as the internal reference gene. The qPCR primers for various cytokines were as follows: MuIL-1β-qF: GCAGCAGCACATCAACAAGAG (SEQ ID NO.14);

[0168] MuIL-1β-qR: CCAGCAGGTTATCATCATCATCC (SEQ ID NO.15); MuIFN-α-qF: GTGAGGAAATACTTCCACAGACTCACT (SEQ ID NO.16); MuIFN-α-qR: TGARGAAGAGAAGGCTCTCATGA (SEQ ID NO.17); MuIFN-β-qF: CAGCACATCTTCGGCATTCTC (SEQ ID NO.18); MuIFN-β-qR: GACGATTCATCTGCCCATAG (SEQ ID NO.19); MuIL-6-qF: GAGCCCACCAAGAACGATAGTC (SEQ ID NO.20); MuIL-6-qF: TCCTCTGTGAAGTCTCCTCTCC (SEQ ID NO.21); MuIL-10-qF: GTTGCCAAGCCTTATCGGAAATG (SEQ ID NO.22); MuIL-10-qR: CTCTTCACCTGCTCCACTGC (SEQ ID NO.23); MuIFN-γ-qF: CTCAAGTGGCATAGATGTGGAAG (SEQ ID NO.24); MuIFN-γ-qR: ACGCTTATGTTGTTGCTGATGG (SEQ ID NO.25); MuIL-4-qF: GTTGTCATCCTGCTCTTCTTTCTC (SEQ ID NO.26); MuIL-4-qR: GGTGTTCTTCGTTGCTGTGAG (SEQ ID NO.27);

[0169] The software GraphPad Prism software version 7.0 was used to perform statistical comparative analysis and draw statistical graphs for the data of each group. The data were expressed as mean ± standard deviation (Mean ± SD). p > 0.05 indicated no significant difference (nosignificant difference, ns), *p < 0.05, **p < 0.01, ***p < 0.001 indicated significant differences.

[0170] Results: In this experiment, the mRNA levels of multiple cytokines in the lung tissues of mice in each group at different infection times were detected by qPCR. The results were as follows Figure 11As shown, for the group infected with BPIV3 alone, the detected mRNA levels of cytokines all showed a trend of first increasing and then decreasing, with significant upregulation at 24 and 36 h, and a positive correlation with the BPIV3 load in the group infected with BPIV3 alone. This indicates that these inflammatory factors all play an immunomodulatory role after BPIV3 infection. For the BPIV3 + TAT - BoIFITM1 and BPIV3 + TAT - BoIFITM2 groups, the mRNA transcription levels of IL - 6, IL1 - β, and IFN - γ were significantly lower than those in the group infected with BPIV3 alone at 24 h and 36 h. The expression levels of IFN - β, IFN - α, TNF - α, IL - 4, and IL - 10 only showed a downward trend at 24 h compared with those in the group infected with BPIV3 alone, and there were no significant differences in the expression levels of other cytokines at other times compared with those in the group infected with BPIV3 alone. This shows that BoIFITM1 and BoIFITM2 play an antiviral role, thus affecting the expression of cytokines and causing their expression levels to rapidly decrease.

[0171] Example 7. Effects of TAT - BoIFITM1 and TAT - BoIFITM2 on the replication of BEV (Bovine enterovirus) or BHV - 1 (Bovine herpesvirus type 1) at the cellular level

[0172] TAT - BoIFITM1 and TAT - BoIFITM2 with a concentration of 0.02 mg / mL were respectively transduced into a 24 - well EBTr cell plate. After 20 min, 1 MOI of BEV or BHV - 1 was added. Cell samples were collected at 6 h, 12 h, 24 h, 36 h, and 48 h respectively, and the viral loads of BEV or BHV - 1 were measured using qPCR.

[0173] The cell samples were washed 3 times with PBS solution, 500 μL of Trizol reagent was added to each well, and after standing at room temperature for 10 min, the cell samples were collected into RNase - free EP tubes. 100 μL of chloroform was added to each tube and shaken vigorously, then left standing at room temperature for 15 min, and centrifuged at 12000 r / min for 15 min using a 4°C centrifuge. At this time, the solution was divided into three layers, and the uppermost layer of the solution was aspirated into a new EP tube, and an equal volume of isopropanol was added and left overnight in a - 20°C refrigerator. Centrifuged at 12000 r / min for 15 min in a 4°C centrifuge, the supernatant was discarded, the precipitate was washed with a pre - prepared 75% ethanol solution, and centrifuged at 7500 r / min for 5 min. The supernatant was discarded, and the ethanol was allowed to dry as much as possible, and the precipitate was dissolved with RNase - free ddH2O. Referring to the reverse transcription kit Plus All - in - one 1st Strand cDNA Synthesis SuperMix to reverse - transcribe RNA into cDNA.

[0174] Reaction procedure: A two-step method was adopted in this experiment. Pre-denaturation was carried out at 95°C for 30 s; denaturation was at 95°C for 5 s, annealing was at 60°C for 34 s, and this cycle was repeated 40 times. After the reaction procedure was completed, the respective gene copy numbers and the corresponding internal reference gene (GAPDH) copy numbers in the test samples were obtained, and the viral load was calculated using 2 -△Ct method. If the calculated result is small, the value can be expanded 1000 times for subsequent analysis and comparison. The software GraphPad Prism software version 7.0 was used to perform statistical comparison analysis and draw statistical charts for each group of data. The paired two-way ANOVA method was used for the significant difference analysis. p < 0.05 was considered statistically significant.

[0175] Results: After transducing EBTr cells with 0.02 mg / mL of TAT-BoIFITM1 and TAT-BoIFITM2 respectively, the cells were infected with BPIV3 at 1 MOI. At 6, 12, 24, 36, and 48 h after infection, the RNA of each group of cells and supernatants was collected to detect the BPIV3 load. The qPCR results showed that after transduction with TAT-BoIFITM1 and TAT-BoIFITM2, the BPIV3 loads inside and outside BBEC and EBTr cells were significantly reduced, especially at 24 h and 36 h, and it was particularly significant at 24 h and 36 h. Moreover, the inhibitory effect of TAT-BoIFITM2 was stronger than that of TAT-BoIFITM1, as Figure 12 shown.

[0176] The experimental results showed that at a concentration of 0.02 mg / mL, TAT-BoIFITM1 and TAT-BoIFITM2 had an obvious inhibitory effect on the replication of BPIV3, and the inhibition rate reached 31% compared with the control group.

Claims

1. A nucleic acid molecule of a bovine IFITM1 gene or a bovine IFITM2 gene, characterized in that: The nucleotide sequence of the bovine IFITM1 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the bovine IFITM2 gene is shown in SEQ ID NO.

6.

2. Use of bovine IFITM1 protein or bovine IFITM2 protein in the preparation of a drug for treating or preventing diseases caused by infection with bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1, characterized in that: The sequence of the bovine IFITM1 protein is shown in SEQ ID NO.12; the sequence of the bovine IFITM2 protein is shown in SEQ ID NO.

13.

3. The use according to claim 2, characterized in that: The bovine parainfluenza virus type 3 is type A, type B or type C.

4. Use of bovine IFITM1 protein or bovine IFITM2 protein in the preparation of a drug for preventing latent infection of bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

5. Use of a recombinant vector carrying bovine IFITM1 protein or bovine IFITM2 protein in the preparation of a drug for treating or preventing bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

6. The use according to claim 5, characterized in that: The starting vector of the recombinant vector is pET-30a.

7. Use of recombinant microbial cells carrying bovine IFITM1 protein or bovine IFITM2 protein in the preparation of drugs for treating or preventing bovine parainfluenza virus type 3, bovine enterovirus and bovine herpesvirus-1.

8. The use according to claim 7, characterized in that: The recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

9. A method for inhibiting the proliferation of bovine parainfluenza virus type 3, bovine enterovirus or bovine herpes virus type 1 for non-therapeutic purposes, characterized in that: The recombinant peptide chain containing bovine IFITM1 protein or bovine IFITM2 protein is transfected into animal cells to obtain recombinant animal cells, and then the recombinant animal cells are infected with bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type 1 to inhibit the proliferation of bovine parainfluenza virus type 3, bovine enterovirus or bovine herpesvirus type 1.

10. The method according to claim 9, characterized in that The animal cells are BBEC or EBTr cells; the dosage of bovine parainfluenza virus type 3, bovine enterovirus or bovine herpes virus type I infected is 1 MOI; the sequence of the recombinant peptide chain containing bovine IFITM1 protein or bovine IFITM2 protein is shown in SEQ ID NO.1 or SEQ ID NO.2; the dosage of the recombinant peptide chain transfection is 0.01 mg·mL-1.

Images