A method for constructing a mouse infection model of orf virus and application thereof

Abstract

The application discloses a method for constructing an orf virus mouse infection model and application thereof, and relates to the technical field of immune engineering. BABL / C mice aged 3-7 days are selected, and orf virus liquid is orally administered to the BABL / C mice, with 30 muL of the orf virus liquid being administered each time, and the administration is continuously performed for 7 days at a dose of 10-7 TCID50. The vaccine can realize a simple immune route, and greatly reduces the waiting time for immune animals in the evaluation of the orf vaccine effect. Meanwhile, the vaccine can greatly accelerate the related mechanism research after the orf infection of animals.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunological engineering, and more particularly to a method for constructing a mouse infection model of orf virus and application thereof. BACKGROUND

[0002] At present, China's sheep industry has entered a period of rapid development, and various epidemic disease prevention and control work has also been valued by everyone. Orf is prevalent in various sheep (including milk goats, cashmere goats and mutton sheep), and causes serious consequences and brings huge losses; especially the morbidity of newborn lambs can reach 60%, and the mortality can reach 10%. At present, there is still a lack of effective vaccines.

[0003] Animal individual immune experiments of orf vaccine, especially the need to immunize pregnant ewes, because they have a longer estrus, pregnancy cycle, and are more concentrated in autumn every year, resulting in a relatively tight animal immune related experiment, and even after the time is missed, the next cycle needs to be waited for, which consumes a lot of time cost. The existing orf research mainly relies on primary cell culture, in vitro experiment and natural infection case, and lacks stability. Therefore, the personnel of the present research urgently need an orf infected substitute animal as an object of orf immune related experiment.

[0004] And the mouse is the most common model animal in the world, and its related immune related kit and antibody product and technology are mature, and it has a wide range of scientific research application conditions, which provides a possibility for subsequent related research. But natural infection in mice is rare. Controllable mouse model to further explore the pathogenic mechanism of the virus, host response and evaluation of prevention and control strategy.

[0005] Therefore, it is an urgent problem for those skilled in the art to develop a mouse model suitable for orf virus infection research. SUMMARY

[0006] Therefore, the present application provides a method for constructing an orf virus mouse infection model and application thereof.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] A method for constructing an orf virus mouse infection model, 3-7 day old BABL / C mice are selected, and orf virus liquid is orally administered, 30 μL of virus is administered each time, and the administration is continued for 7 days at a dose of 10-7 TCID50.

[0009] Further, the orf virus is orf virus virulent strain ORFV-WF-3, and the preservation number is CGMCC No. 17993, see patent 202110250629.1.

[0010] Further, the mouse is a BABL / C mouse.

[0011] Application of the mouse infection model of orf virus in evaluating pathogenicity of orf virus.

[0012] Application of the mouse infection model of orf virus in evaluating orf virus vaccine.

[0013] Application of the mouse infection model of orf virus in evaluating orf disease drugs.

[0014] The beneficial effects of the application compared with the prior art are known through the above technical solutions.

[0015] The vaccine can realize a simple immunization route, greatly reduces the waiting time of the immune animals in the evaluation of orf vaccine effect, and can greatly accelerate the related mechanism research after the orf infection of animals BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of the provided drawings.

[0017] Figure 1 Fluorescence quantitative detection results of orf virus in blood tissue;

[0018] Figure 2 Results of body weight changes of young mice after orf virus infection;

[0019] Figure 3 Results of orf virus group identification in intestinal tissue;

[0020] Figure 4 Results of PCR detection in blood after immunization;

[0021] Figure 5 Results of virus titer changes in blood after immunization;

[0022] Figure 6 Results of virus carrying in different tissues after orf virus challenge;

[0023] Figure 7 Results of virus titer detection in intestinal tissue after immunization;

[0024] Figure 8 Results of cytokine level detection in intestinal tissue after immunization. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort fall within the protection scope of the present application.

[0026] Embodiment 1

[0027] 1 Preparation of host cells:

[0028] The frozen goat testicular cells GT-26 were taken out from the working cell bank, thawed in a 37℃ water bath, centrifuged at 1000-1500 r / min for 5-8 min, and the supernatant was discarded. After resuspension in MEM medium containing 8-10% newborn calf serum, 25 cm 3 cell bottles were inoculated, and the cell bottles were cultured in a 37℃ incubator containing 5% CO2 to a dense monolayer. The cells were digested with 0.25% trypsin, subcultured at a ratio of 1:3-4, and expanded. The expanded goat testicular cells GT-26 were inoculated into cell transfer bottles and cultured at 37℃ to a dense monolayer at a speed of 8-11 r / h;

[0029] 2 Virus transfer bottle culture and harvesting:

[0030] The goat testicular cells GT-26 with a good monolayer in the transfer bottle were discarded, and ORFV-WF-3 was added at a ratio of 1% (V / V). MEM medium was added to the transfer bottle at a volume of 10%, and the transfer bottle was cultured at 37℃ in a warm room at a speed of 8-11 r / h. The cytopathic effect was observed every day, and the virus liquid was harvested after 48-72 h of culture and stored at -20℃ or below;

[0031] 3 Preparation of an orf mouse infection model:

[0032] BABL / C mice aged 3-7 days were selected, and the ORFV experimental group was orally infected with ORFV virus liquid, 30 μL each time, at a dose of 10-7 TCID50 for 7 days. The detection was performed after 7 days of interval;

[0033] 4 Detection of the orf mouse infection model:

[0034] The mice were dissected, and blood, intestinal tract, mesenteric lymph nodes, liver, spleen and other tissues were collected. PCR was used for molecular level detection of the ORFV pathogen;

[0035] 5 Application of the orf mouse infection model in orf vaccine immunization

[0036] The pregnant female mice in the experimental group were subcutaneously immunized with orf virus inactivated vaccine (produced by Haerbin) twice, 0.1 ml / time, with an interval of 2 weeks, and the control group was subcutaneously injected with normal saline twice, 0.1 ml / time, with an interval of 2 weeks. After the mice gave birth, the pups were given orf virus secondary challenge 30 μL orally, with an amount of 10-7 TCID50 for 7 days, and the change of orf virus was detected after an interval of 7 days.

[0037] (I) Preparation of orf virus mouse infection model, the steps are as follows:

[0038] Select 3-day-old BABL / C mice, and orally administer orf virus liquid to the experimental group, 30 μL each time, with an amount of 10-7 TCID50 for 7 days, and then measure the body weight after an interval of 7 days; the control group is orally administered with an equal amount of normal saline;

[0039] Dissect the mice, collect blood, intestinal tract, mesenteric lymph nodes, liver, spleen, and other tissues, use PCR to detect the molecular level of orf virus pathogens, use RT-PCR to determine the load of orf virus in blood, and use intestinal tissue immunohistochemical sections to finally determine whether the virus can infect intestinal tissue and peripheral blood.

[0040] (II) Identification and detection method of orf virus mouse infection model

[0041] 1) Tissue orf virus PCR detection

[0042] 1.1 Extraction of nucleic acid from peripheral tissue and fluorescence quantitative detection

[0043] Use an animal tissue genomic DNA extraction kit to extract total DNA from the peripheral blood of mice, determine the nucleic acid concentration (OD 260 / OD 280 ), and then perform fluorescence quantitative PCR (Real-time PCR) detection.

[0044] The amplification system is 20 μL: 2×PerfectStart Green qPCR SuperMix 10 μL;

[0045] Primer sequence:

[0046] Orfv-b2l F: 5'-ATCACCGAGTGGAAGAACGC-3', as shown in SEQ ID NO. 1;

[0047] Orfv-b2l R: 5'-CGATGAGCAGCTTGGTGTTG-3", as shown in SEQ ID NO. 2;

[0048] Upstream and downstream primers 0.4 μL each, DNA template 2 μL, ddH2O (RNase-free) 7.2 μL. Amplification conditions (full type two-step method): 94°C 30 s; 94°C 5 s, 60°C 30 s, 45 cycles.

[0049] Results are shown in Figure 1 The detection of ORFV in blood tissue showed that the sheep pox virus entered the mouse body and achieved the purpose of infecting the mouse.

[0050] 2) Weight detection of infected model animals

[0051] The pathogenicity of ORFV to mice was observed every day after the challenge test, and the clinical manifestations and weight changes of the mice were monitored. The results are shown in Figure 2 The body weight of the mice in the experimental group decreased significantly, and the statistical analysis showed that there was a significant difference in the body weight changes of the mice in the two groups.

[0052] 3) Antibody staining of tissues of infected model animals

[0053] 3.1 Detection of sheep pox in tissues at the infection site of the tissue

[0054] (1) Tissue processing: Intestinal tissue was taken and preserved in 10% formalin

[0055] Fixation and dehydration: The fixed tissue was dehydrated, transparent, and immersed in wax, etc. to prepare paraffin-embedded blocks.

[0056] Slicing: The paraffin block was cut into thin slices about 4-6 microns thick and attached to a glass slide.

[0057] (2) Antigen repair and blocking

[0058] Dewaxing and hydration: The section was dewaxed with xylene and then gradually hydrated by alcohol gradient.

[0059] Antigen repair: Methods such as heating and enzyme digestion were used to restore the epitope of the antigen to improve the binding efficiency of the antigen and the antibody.

[0060] Endogenous peroxidase blocking: The section was treated with a solution containing hydrogen peroxide to eliminate the activity of endogenous peroxidase and avoid non-specific staining.

[0061] (3) Incubation of primary antibody

[0062] The primary antibody (rabbit anti-ORFV monoclonal antibody) specific to the antigen of ORFV was used to incubate at an appropriate temperature for a certain period of time to allow the primary antibody to bind to the target antigen.

[0063] (4) Secondary antibody and color development

[0064] Secondary antibody incubation: add biotin-labeled secondary antibody, incubate at room temperature for one hour Color / fluorescence detection: use the corresponding substrate DAB to develop color, so that the bound secondary antibody can be seen, thus showing the situation of orf virus infected cells.

[0065] Results are shown in Figure 3 In the infected group of histochemical detection, the presence of orf virus infected cells in the tissue can be observed, and the control group does not exist, indicating that orf virus has infected the intestinal tissue

[0066] (III) Application of orf mouse infection model

[0067] 1) Application of immune effect of orf vaccine in mouse model

[0068] Pregnant female mice (3 pregnant mice in each group) were immunized subcutaneously with orf inactivated vaccine (produced by Harbin Pharmaceutical Group) 2 times, 0.1 ml / time, with an interval of 2 weeks, and the control group was injected subcutaneously with normal saline 2 times, 0.1 ml / time, with an interval of 2 weeks. After the mice gave birth, the pups were given orf virus secondary challenge 30 μL orally 5 days later, with a dose of 10-7 TCID50 for 7 days. After the challenge, the pups were dissected 7 days later, and the blood was collected for PCR detection of ORFV pathogens at the molecular level. The primer sequence and method are described in "Research on IL-17-mediated immune pathological damage of goat orf" by Tang Qidan, 2020, Northwest A&F University; the target fragment size is 560 bp, and the change of orf virus in blood is detected by fluorescence quantitative.

[0069] 3.1.1 Detection of ORFV pathogens at the molecular level by PCR

[0070] Results are shown in Figure 4 In the control group, the blood tissue ORFV detection found that orf virus fragments were detected in the blood of mice after challenge, indicating that orf virus entered the mouse body, while the experimental group did not detect orf virus, indicating that after immunization with orf vaccine, orf virus infection can be effectively prevented.

[0071] 3.1.2 Change of orf virus in blood

[0072] 1.1 Extraction of peripheral blood nucleic acid and fluorescence quantitative detection

[0073] Total DNA in the peripheral blood of mice was extracted using an animal tissue genomic DNA extraction kit, and the nucleic acid concentration (OD260 / OD280) was determined before fluorescence quantitative PCR (Real-time PCR) detection.

[0074] The amplification system is 20 μL: 10 μL of 2x PerfectStart Green qPCR SuperMix, 0.4 μL of each of the upstream and downstream primers, 2 μL of DNA template, and 7.2 μL of ddH2O (RNase-free). The amplification conditions (full type two-step method) are: 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, 45 cycles.

[0075] The results are shown in Table 1. Figure 5 In the immunization group, no virus was detected, while in the control group, the orf virus was detected in the mouse at different times. It can be found that the virus titer reaches the highest 105cops at 2 days after infection, and then gradually decreases to 104cops after 2 weeks. At the same time, it also shows that after immunization with orf vaccine, the infection of orf virus can be effectively prevented

[0076] Example 2

[0077] 1. Preparation of orf mouse infection model:

[0078] Select 7-day-old BABL / C mice, and orally administer ORFV virus liquid to the experimental group, 30 μL each time, at a dose of 10-7TCID50, for 7 days. After an interval of 7 days, detection is performed.

[0079] 2. Detection of orf mouse infection model:

[0080] Dissect the mouse, collect blood, intestinal tract, mesenteric lymph nodes, liver, spleen and other tissues, and use PCR to detect the molecular level of ORFV pathogen;

[0081] The results are shown in Table 1. Figure 6 In the infected mouse tissues (blood, intestinal tract, mesenteric lymph nodes, liver, spleen), the same size fragment as the target gene can be observed, indicating that orf virus exists in these tissues, and that after infection, these tissues have orf virus-infected cells.

[0082] 3. Application of orf mouse infection model in orf vaccine immunization

[0083] Pregnant female mice, 3 per group, were subcutaneously immunized with orf inactivated vaccine (produced by Harbin Pharmaceutical Group) twice, 0.1 ml each time, with an interval of 2 weeks, and the control group was subcutaneously injected with physiological saline twice, 0.1 ml each time, with an interval of 2 weeks. After the mice gave birth, the pups were orally administered orf virus 7 days later, 30 μL each time, at a dose of 10-7TCID50, for 7 days. After an interval of 7 days, the cytokine level in the intestinal tissue was detected.

[0084] (II) Identification and detection method of orf virus mouse infection model

[0085] 1) Detection of orf virus and cytokines in intestinal tract

[0086] 1.1 Tissue nucleic acid extraction and detection of orf virus by qPCR

[0087] The total DNA of mouse intestinal tract was extracted by animal tissue genomic DNA extraction kit, and the nucleic acid concentration (OD260 / OD280) was determined, and then Real-time PCR was performed. The amplification system was 20 μL: 2x PerfectStart Green qPCR SuperMix 10 μL, upstream and downstream primers (Orfv-b21 F and Orfv-b21 R) 0.4 μL each, DNA template 2 μL, ddH2O (RNase-free) 7.2 μL. The amplification condition (full type two-step method): 94°C for 30 s; 94°C for 5 s, 60°C for 30 s, 45 cycles.

[0088] The results are shown in Figure 7 The detection of ORFV in the intestinal tissue of the control group found that orf virus was detected in the intestinal tract of mice after challenge, indicating that orf virus entered the intestinal tissue of mice, and the same size fragment of orf virus was not detected in the experimental group, indicating that the mice were not infected with orf virus, and vaccine immunization could effectively prevent the infection of orf virus in the intestinal tissue.

[0089] 1.2 PCR detection of orf virus and cytokine detection

[0090] The cytokines (IL-1β, IL-6, IFN-γ) were detected by fluorescence quantification, and the primer sequences are shown in Table 1.

[0091] Table 1: Fluorescence quantitative detection of cytokines

[0092]

[0093]

[0094] GAPDH was used as an internal reference gene as a blank.

[0095] The results are shown in Figure 8 By comparing the detection results of cytokines, it was found that IL-6 and IL-1β and IFN-γ were increased to different degrees, and the increase of IL-6 and IL-1β inflammatory cytokines indicated that the mouse body had inflammatory reaction, and the increase of IFN-γ indicated that humoral immunity should play a role in the infection of orf virus, and could quickly clear the virus existing in the circulatory system of the body.

[0096] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a mouse model of poxvirus infection, characterized in that, BABL / C mice aged 3-7 days were selected and orally administered 30 μL of ovine pox virus solution each time for challenge. -7 TCID 50 The amount of virus was continuously attacked for 7 days; The sheep pox virus is a virulent strain of sheep pox virus, ORFV-WF-3, with the accession number CGMCC No. 17993.

2. The application of the mouse infection model of orthopnea virus obtained by the construction method of claim 1 in evaluating the pathogenicity of orthopnea virus.

3. The application of the mouse infection model of orthopnea virus obtained by the construction method of claim 1 in evaluating orthopnea virus vaccines.

4. The application of the mouse infection model of orthopnea virus obtained by the construction method of claim 1 in evaluating drugs for orthopnea disease.

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