CN113429477A - Preparation method and application of novel coronavirus, specific antibody-containing milk or serum produced by immune dairy cow thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method and application of a novel coronavirus and specific antibody-containing milk or serum produced by an immune cow thereof. A large amount of immune milk and serum can be obtained by utilizing the inactivated novel coronavirus SARS-CoV-2/ZY38-1 to immunize a cow, and both the immune milk and the serum contain high-content antibodies, so that the virus can be effectively neutralized, the virus invasion cells can be prevented, and the SARS-CoV-2 virus resistance effect is obvious; after the Igs protein purified by the immune milk is injected into a mouse, the death rate of the mouse infected by the new coronavirus can be obviously reduced, the influence of the new coronavirus infection on the weight reduction of the mouse is reduced, and the virus content in the lung tissue of the mouse is reduced. The immune milk and the serum provided by the invention can be used as antibody sources of a new coronary pneumonia prevention and treatment preparation, and make up for the limitation of the source of new coronary pneumonia rehabilitation plasma.

Description

Preparation method and application of novel coronavirus, specific antibody-containing milk or serum produced by immune dairy cow thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of positive milk and serum of severe acute respiratory syndrome coronavirus type 2 and specific antibodies thereof.

Background

A novel coronavirus pneumonia (coronavirus disease 19, COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) appeared at the end of 2019 and presented a global outbreak, seriously endangered human health and public health safety, brought huge burden to world society and economy, and caused high attention globally. By 7 months in 2020, the total number of confirmed cases of new coronary pneumonia exceeds 1400 million and the number of deaths exceeds 60 million. Many foreign and domestic scholars speculate that new coronavirus or similar influenza virus will coexist with human for a long time. Moreover, the existing research reports and data show that the fatality rate of SARS-CoV-2 is much higher than that of influenza, but no therapeutic and prophylactic preparation or vaccine specifically aiming at resisting novel coronavirus can be used so far, so that an effective prevention and treatment method aiming at SARS-CoV-2 is urgently needed to be found. Inhibition of viral entry into cells is a very effective therapeutic strategy. Studies have reported that angiotensin converting enzyme 2 (ACE 2) is a functional receptor for SARS-CoV, and is capable of binding with high efficiency to S protein on the surface of SARS-CoV, which invades cells via the cell surface receptor ACE2 (Li Wenhui et al, 2003; Kuba K, 2005). Therefore, researchers have constructed recombinant ACE2 protein, which was found to have potential therapeutic effects on SARS-CoV (Kuba K, 2005). Daniel W et al (2020) reported that the S protein of SARS-CoV-2 also binds to the ACE2 receptor with much higher affinity than the S protein of SARS-CoV. Vanessa Monteil et al (2020) study reports that when cells are infected with SARS-CoV-2 and incubated with recombinant human ACE2 protein purified in vitro for a period of time, the infectivity of the virus is rapidly reduced, and the reduction range can reach 1000-5000 times, and the study also finds that the infection of S ARS-CoV-2 cannot be inhibited when cells are infected with SARS-CoV-2 and incubated with murine ACE2 protein for a period of time. However, it was found that recombinant rACE2 had a half-life of only a few hours and was cleared quickly in vivo (Wysocki J et al, 2010; Haschke M et al, 2013). Clinically, serum of a rehabilitation patient with the novel coronavirus pneumonia has a certain curative effect on critically ill patients, but a method for treating virus infection by using blood plasma of the rehabilitation patient cannot be popularized, and on one hand, the recovery patient is weak and is not suitable for blood donation, and on the other hand, the recovery patient is limited by factors such as blood plasma sources.

The invention reports that SARS-CoV-2 is put out of fire to immunize milk cow, and the serum and milk after immunization are collected as a special 'antibody' to neutralize SARS-CoV-2 and prevent virus from invading cells for the first time, and the invention is a biological preparation for effective prevention and treatment specially aiming at novel coronavirus infection.

Disclosure of Invention

The invention aims to provide a novel isolated coronavirus, wherein the novel coronavirus strain is delivered to China center for type culture Collection (CGMCC) at 04.01/2021, and is classified and named as follows: SARS-CoV-2/ZY38-1, accession number: CCTCC NO: V202107, address: wuhan university in Wuhan, China.

The present invention aims at solving the problem of severe acute respiratory syndrome coronavirus (SARS-CoV-2) difficult to prevent and treat, the application of new coronavirus strain in preparing positive milk or serum containing new coronavirus antibody, using the prepared antigen to immunize milk cow to produce immune serum and immune milk containing specific antibody for resisting SARS-CoV-2 virus, and using the obtained new coronavirus immune milk to purify Igs protein for preparing biological preparation for treating or preventing SARS-CoV-2 infection.

In order to achieve the purpose, the invention adopts the following technical measures:

an isolated novel coronavirus strain which has been delivered to the China center for type culture Collection at 04.01.2021, under the taxonomic nomenclature: SARS-CoV-2/ZY38-1, accession number: CCTCC NO: V202107, address: wuhan university in Wuhan, China.

The application of the novel coronavirus strain in preparing positive milk or serum containing the specific antibody of the novel coronavirus, wherein the accession number of the novel coronavirus is EPI _ ISL _ 486645; the application process comprises injecting inactivated new coronavirus strain into cow via neck or buttocks muscle to obtain milk or serum containing new coronavirus antibody positive. The novel coronavirus has a deposit number of: CCTCC NO: V202107, address: wuhan university in Wuhan, China.

In the above application, preferably, the inactivated new coronavirus is prepared into an immune preparation to immunize a cow, and the inactivated new coronavirus is centrifuged to remove cell debris, and then concentrated to obtain a high-concentration antigen;

preparation of an aqueous phase: adding 2-4% Tween 80 into the concentrated antigen;

preparing an oil phase: 5-6% of span 85 and 1-2% of aluminum stearate are added into the macro152 white oil, and sterilization is carried out;

and mixing the water phase and the oil phase according to the proportion of 3: 2-4: 3 to obtain the new coronavirus immune preparation.

The protection scope of the invention also includes:

the milk or serum prepared by the method is used for evaluating a novel coronavirus animal model, and the model is constructed by infecting a mouse with SARS-CoV-2/WBP-1.

The milk prepared by the method is prepared into a drinking medicament for neutralizing the novel coronavirus.

The milk or serum prepared by the method can be used for preparing medicines for treating or preventing novel coronavirus infection.

The milk or serum prepared by the method is used for preparing novel coronavirus Igs antibodies, and the antibody protein is used for preparing medicaments for treating or preventing novel coronavirus infection.

Compared with the prior art, the invention has the following advantages:

1. the novel coronavirus can obtain a large amount of immune milk after immunizing a cow, and the immune milk and serum both contain high-content antibodies, can be used as an antibody source of a preparation for preventing and treating new coronary pneumonia, makes up for the limitation of the source of recovered plasma of the new coronary pneumonia, and can be more widely applied to the new coronary pneumonia caused by the novel coronavirus.

2. The immune serum and the immune milk contain high-concentration specific antibody aiming at SARS-CoV-2, can effectively neutralize virus, prevent the virus from invading cells, and have obvious effect of resisting SARS-CoV-2 virus.

3. After the Igs antibody purified by the novel coronavirus immune milk is injected into a mouse, the death rate of the mouse infected by the new coronavirus can be remarkably reduced, the influence of the new coronavirus infection on the weight reduction of the mouse is reduced, and the virus content in the lung tissue of the mouse is reduced.

Drawings

FIG. 1 Western-blot assay of RBD protein expression.

FIG. 2 immunomilk and serum samples New coronavirus ZY38-1 neutralizing antibody titer plaque plots.

FIG. 3 is a schematic representation of the effect of immune milk on the body weight of SARS-CoV-2/WBP-1 virus infected mice.

FIG. 4 is a schematic representation of the effect of immune milk on the survival of mice infected with SARS-CoV-2/WBP-1 virus.

FIG. 5 is a schematic diagram showing the effect of immune milk on the viral load of SARS-CoV-2/WBP-1 virus infected mouse lung tissue.

Detailed Description

Unless otherwise specified, the test methods and conditions in the examples of the present invention are conventional methods. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. The technical scheme of the invention is a conventional scheme in the field if no special description is provided; the reagents or materials are commercially available, unless otherwise specified. All tests related to the live SARS-CoV-2 virus were performed in the biosafety third-level laboratory (ABSL 3).

Example 1:

preparation of milk and serum containing novel coronavirus antibodies:

the sequence of the novel coronavirus strain is listed in a GISAID database submitted to No. 7/10 of 2020, the login number is EPI _ ISL _486645, and the preservation number in the China center for type culture Collection is CCTCC NO: V202107; in the present invention, the virus is simply referred to as novel coronavirus ZY 38-1.

1. Proliferation culture and inactivation of novel coronavirus ZY38-1

(1) Cell culture:

VERO cells are cultured by DMEM containing 10% FBS for about 48 hours for passage;

cell passage: when in passage, the mixture is digested by 0.25 percent of pancreatin for about 10 minutes.

The method comprises the following specific steps:

sucking old culture solution in a culture vessel by using a dropper or a pipette, and washing residual old culture medium by using a DMEM (DMEM) culture medium without serum;

adding 1-2mL of digestive juice (0.25% pancreatin) into the flask, and gently shaking the flask to make the digestive juice flow over all cell surfaces;

when the cells are withdrawn and the processes become round or the intercellular spaces increase, the digestion should be terminated immediately (the digestion solution is aspirated or poured off, a small amount of fresh culture solution containing serum is added to terminate the digestion) under an inverted microscope;

sucking the culture solution in the bottle by using a pipette, repeatedly blowing and beating the digested cells to enable the digested cells to be detached from the wall and dispersed to form cell suspension;

counting, subpackaging into new culture bottles, and supplementing a certain amount of fresh culture solution containing serum;

covering with bottle cap, screwing up, and slightly rotating for CO₂Introducing gas, and returning CO to the culture flask₂An incubator.

(2) Proliferation of novel coronavirus ZY 38-1:

inoculating the digested Vero cells into a cell culture bottle, and placing the cell culture bottle at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator until a monolayer of cells grows out;

inoculating-80 deg.C-preserved novel coronavirus ZY38-10.1mL into culture flask with single layer Vero cells, and culturing at 37 deg.C with 5% CO₂Incubating for 1h in a constant-temperature incubator;

carefully aspirate the virus incubation by pipette, add 10mL of DMEM cell maintenance medium containing 2% FBS, 5% CO at 37 deg.C₂Culturing in a constant temperature incubator for 48-72 h;

observing the cell state every day, and harvesting virus cell proliferation liquid when more than 80% of cells have lesions; the obtained new coronavirus solution has a virus titer of 10^6.5TCID50/mL。

(3) Inactivation of SARS-CoV-2 virus and verification of inactivation effect:

virus inactivation: the new coronavirus propagated by the cells is inactivated twice by beta-propiolactone. The specific method is that one thousandth of beta-propiolactone is inactivated at 4 ℃ for 24 hours, and the beta-propiolactone is degraded at 37 ℃ for 6 hours.

And (3) inactivation effect verification: and inoculating the inactivated virus to Vero, observing for 70 hours, collecting supernatant, performing blind transmission for three generations, and preventing cells from generating lesions. Further, the inactivated virus solution is inoculated to a mouse in an abdominal cavity, and the mouse is observed for 70 hours without any adverse reaction, which indicates that the inactivation is thorough and can be used for the next step of antigen preparation.

2. Novel coronavirus antigen preparation

And (3) centrifuging the inactivated and qualified new coronavirus liquid to remove cell debris, and concentrating by adopting a membrane with the aperture of 100KD for 20 times to obtain the high-concentration antigen.

Preparation of an aqueous phase: to the concentrated antigen was added 2% tween 80.

Preparing an oil phase: adding 6% span 85 and 1% aluminum stearate into macro152 white oil, and sterilizing at 116 deg.C for 30 min.

And mixing the water phase and the oil phase according to the proportion of 4:3 to obtain the neocoronavirus oil emulsion immune antigen.

3. Prepared coronavirus oil emulsion antigen immune milk cow

Animal immunization: the prepared antigen is injected into the neck and hip muscles of the dairy cattle for immunization, 16 cattle are immunized, 6mL of the antigen is immunized in each cattle, the immunization is carried out once every 14 days, the immunization is carried out for 4 times totally, and no adverse reaction occurs in the immunized cattle.

Collecting and processing samples: blood samples and milk samples of the immunized cows are collected at different time points after immunization respectively to carry out novel coronavirus antibody detection. The blood sample collection adopts a tail vein blood collection method, a disposable syringe is used for aseptically collecting 3-5mL of non-anticoagulated blood, the blood sample is kept still to separate out serum, the blood sample can not be shaken violently to avoid hemolysis, the blood sample is sent to a laboratory to be separated into serum immediately, and the blood sample which can not be separated immediately needs to be refrigerated in time (4 ℃).

Example 2:

determination of specific antibody levels in immune milk and immune serum

1. Expression and purification of novel coronavirus RBD antigen protein

Through the design of expression elements and the reconstruction of vectors, a novel coronavirus RBD gene is inserted into a pCMV3 expression vector to construct a pCMV3-RBD-his eukaryotic expression vector, and a target protein is obtained through transient expression in 293T suspension cells. 1.1 construction of pCMV3-RBD-his plasmid

Extracting inactivated ZY38-1 virus RNA and reverse transcription amplification virus cDNA, using the cDNA template for RBD fragment amplification, and amplifying a primer RBD-F: 5'-ATGAGGGTCCAACCAACAGAGAGC-3'; 5'-GAAGTTCAC ACACTTGTTCTTCAC-3' is RBD-R. In order to facilitate the subsequent purification of RBD protein, a 6 XHIS sequence is added at the rear end of the RBD gene to obtain the RBD-HIS gene. And carrying out double enzyme digestion on the pCMV3 empty vector and the RBD-his gene by using KpnI and XbaI, and connecting the enzyme-digested fragment with the vector to obtain a recombinant plasmid. The recombinant plasmid is transformed into escherichia coli DH5a competent cells, and cloning screening and sequencing identification are carried out to obtain the correct recombinant plasmid pCMV 3-RBD-his. Carrying out amplification culture on Escherichia coli with positive pCMV3-RBD-his plasmid, extracting non-endotoxin pCMV3-RBD-his plasmid by using MN plasmid extraction kit, adjusting the concentration of the extracted plasmid to 1 ug/mu L, and subpackaging at-80 ℃ for storage.

1.2 recovery and culture of suspension 293T cells

The 293T suspension cell frozen stock solution is thawed in a water bath at 37 ℃,20 mL of culture medium is added into a 100mL cell shaking flask, all the thawed cells are transferred into a cell flask, and the cells are gently mixed. Culturing the resuspended cells in a constant-temperature shaking table with the rotating speed of 150-175rp m at 37 ℃ under 5% CO2, detecting the cell activity within 3-5 days, carrying out passage for 2-3 times, and carrying out transfection when the cell activity is more than 90%.

1.3 transfection of cells

Taking 20 mu L of the pCMV3-RBD-his plasmid preserved in the step 1.1 to 150mmol of NaCl solution, wherein the final volume is 1mL, and the plasmid content is 20 ug; meanwhile, diluting 100 mu L of transfection reagent by using 150mmol of NaCl to the total volume of 1mL, standing alone for five minutes, mixing the 2 solutions uniformly, and dropwise adding the mixed solution into the shake flask of cells with the cell activity of more than 90%. And (5) detecting the expression quantity of the target protein in the cells and the supernatant after transfection for 48-72 h.

1.4 expression product analysis

Transfecting 293T cells with pCMV3-RBD-his, collecting protein sample samples in cultured cells and supernatant at four time points of 24h, 36 h, 48 h and 72h, purifying target protein by using a nickel column, and carrying out Western-blot detection to prove that the RBD protein starts to express at 24h and the RBD protein content in the cultured supernatant is highest at 72h, as shown in figure 1.

Determining the concentration of the purified RBD protein by using a Bradford protein concentration determination kit, diluting the obtained RBD protein by 50 times by using a coating solution, coating a 96-well polystyrene reaction plate by 100 mu L per well, and incubating overnight at 2-8 ℃; removing the coating solution, adding sealing solution, sealing at 37 deg.C for 40 min; discarding the blocking solution, washing with PBST for 3 times, diluting RBD protein specific labeled monoclonal antibody 1000 times with diluent (5% defatted milk powder PBST), adding 100 μ L per well, and incubating at 37 deg.C for 40 min; sucking dry, washing with PBST for 3 times, adding 100 μ L TMB working solution into each well, keeping away from light for 10min, adding 50 μ L stop solution, and measuringDetermining OD₆₃₀And (4) light absorption value. The result shows that the RBD protein after purification has specific ELISA reaction.

2. ELISA detection steps of specific antibodies of the novel coronavirus ZY 38-1:

(1) and (3) taking the novel coronavirus RBD protein expressed and purified in the step (1) as an antigen, coating a 96-hole polystyrene reaction plate by using 100 ng/hole, incubating at 4 ℃ for 16h, removing a coating solution, drying an ELISA coating plate, adding a sealing solution, and sealing at 37 ℃ for 2 h. Removing the blocking solution, beating dry the ELISA coated plate, adding 180 muL of PBST washing solution into each hole, washing for 3 times, beating dry the ELISA coated plate, drying, and placing at 4 ℃ for later use.

(2) Centrifuging the blood sample to be detected at 4 deg.C and 3000rpm for 10min, centrifuging the milk sample to be detected at 4 deg.C and 8000 rpm for 20min, collecting the intermediate layer, and storing at-20 deg.C.

(3) The stored serum and milk samples were diluted 1:40 with a diluent (probiotics, GmbH, Wuhan Ke.), and added to the antigen-coated plate at 100. mu.L/well. Incubating in 37 ℃ incubator for 30min, discarding coating solution, adding 180 μ L PBST washing solution to each well, washing for 5 times, and beating dry ELISA coated plate.

(4) Diluting rabbit anti-bovine IgG antibody labeled by horseradish peroxidase to 1:16000 with a secondary antibody protective agent (limited pre-biological member of the Wuhan family), adding into an ELISA coated plate incubated with the primary antibody, incubating for 30min at 37 ℃ in an incubator at 100 mu L/hole, discarding secondary antibody diluent, adding 180 mu L of PBST washing solution into each hole, washing for 5 times, and patting dry the ELISA coated plate.

(5) 50. mu.L of TMB color developing solution A, 50. mu. L, B, was added to each well, and the reaction was stopped by keeping the wells away from light at 37 ℃ for 10min and then adding 50. mu.L of stop solution to each well.

(6) The reaction plate after termination is placed in a microplate reader, and the OD of each well is read_630nmAnd (4) data.

As shown in tables 1 and 2, the antigen prepared from SARS-CoV-2 virus can stimulate host to produce antibodies against the novel coronavirus after immunizing cow, and can detect the novel coronavirus antibodies in both the serum and the milk of immunized cow, and the novel coronavirus antibodies can still be detected in the milk 76 days after the fourth immunization, and the serum sample also contains high antibody level.

TABLE 1 SARS-CoV-2 specific antibody levels in immune milk

Note: "-" indicates that the cow was in the dry period and no sample was taken.

TABLE 2 SARS-CoV-2 specific antibody levels in immune sera

Note: "-" indicates that the cow was in the dry period and no sample was taken.

Example 3:

neutralizing antibody titer of immune milk and immune serum

The method adopts a trace neutralization experiment for reducing plaque of the new coronavirus to detect the titer of a neutralizing antibody of an antibody in immune milk and immune serum to the new coronavirus, and comprises the following specific steps:

(1) the Vero cells with good growth state are subcultured to a 12-hole cell culture plate with 3-5E 5/hole, and grow into a compact single layer the next day.

(2) Serum and milk samples treated and stored at-20 ℃ in example 2 were inactivated for 30min at 56 ℃.

(3) Serum and milk samples were diluted in 24-well plates in serum-free DMEM medium at double ratios, and 24-well plates were prepared for the number of samples, 4 samples per plate, and an additional virus back-drop control was added.

(4) The first column of each well plate was loaded with 441. mu.L or 405. mu.L of DMEM, and the other wells were loaded with 300. mu.L.

(5) Add 9. mu.L of serum or 45. mu.L of milk samples to the first row of wells, i.e.the total volume of liquid in each well was 450. mu.L, mix well with shaking.

(6) Taking 150 mu L from the first row to the second row by using a row gun, blowing, beating, vibrating and uniformly mixing; the tip was changed, 150. mu.L was taken from the second row to the third row, the operation was repeated until the sixth row, and finally 150. mu.L was taken from the sixth row and discarded.

(7) And calculating the volume of the required virus dilution liquid and virus freezing liquid according to the number of samples, adding 3-5mL of dilution liquid on the basis of the required volume, and taking DMEM + 2.5% FBS with the corresponding volume into a 50mL centrifugal tube.

(8) A frozen stock of the novel strain of coronavirus ZY38-1 was removed from a freezer at-150 deg.C, inserted into the buoy as soon as possible and placed in a box filled with tap water, and the buoy was removed from time to accelerate thawing.

(9) Taking the virus freezing medium with the required volume to DMEM diluent, wherein the virus titer after dilution is 400 pfu/mL; if the volume of the required virus frozen stock solution is less than 10 mu L, the virus frozen stock solution is firstly diluted by 10 times in a 2mL centrifugal tube.

(10) And (3) taking 300 mu L of virus diluent and adding the virus diluent into serum or milk diluent, and changing the gun head for each diluent to avoid the virus from not acting with the serum or milk due to collision with the hole wall.

(11) Adding virus diluent, shaking each 24-well plate, mixing, placing in incubator at 37 deg.C for 1h, and stacking the plates as little as possible, or stacking more than two layers if the space is limited.

(12) Absorbing the culture medium in the Vero cell culture plate, and adding 500 mu L of serum or mixed solution of milk and virus; incubate at 37 ℃ for 1h in an incubator with as little plates stacked as possible, if space is limited, no more than two layers stacked.

(13) The mixture was aspirated off, 1mL of DMEM + 2.5% FBS + 0.9% methylcellulose was added to each well, and the mixture was placed in an incubator for 3-4 days.

(14) Add 1mL of 8% formaldehydee-PBS to each well and fix for more than 1 h.

(15) The fixative was poured off and washed once with single distilled water.

(16) Adding 0.5% crystal violet for dyeing for 10min, washing with single distilled water for three times, and taking pictures and counting plaques.

As a result, as shown in tables 3 and 4, both the immune serum and the immune milk were effective in inhibiting the proliferation of the novel coronavirus ZY38-1 on cells. After 14 days of four-time immunization, the neutralizing antibody titer of the immune bovine serum reaches 1: 12150; the neutralizing antibody titer in the immune milk can reach 1:270 by more than 85 percent.

TABLE 3 neutralization potency of SARS-CoV-2 specific antibodies in immune milk

TABLE 4 neutralization potency of SARS-CoV-2 specific antibodies in immune sera

Example 4:

evaluation of antiviral effect of novel coronavirus ZY38-1 immune milk in mice:

obtaining mouse adaptive strain SARS-CoV-2:

SARS-CoV-2 strain (NC-045512.2) separated from human sample is inoculated into female B ALB/c mouse of 10 months age by nasal drip, the infection dose is 50ul, the virus content is 2.3X 10⁵PFU. After 3 days of infection, the mice were dissected to collect lung tissue, homogenized, centrifuged at 12000rpm for 5min, and the supernatant was collected as mouse-adapted passage new coronavirus passage 1. The lung tissue homogenate of the 1 st generation was inoculated into 4-6 week old mice, and 3 days later, the lungs were harvested and homogenized to obtain virus fluid of the 2 nd generation. The mice aged 4-6 weeks were serially passaged to obtain 11 th generation virus solution. During the passage, the 9 th mouse died due to virus infection for the first time, and the mice died after passing through 14 generations.

Homogenizing the supernatant of the 11 th lung tissueDilution 10³、10⁴Inoculating the strain to VERO cells in a doubling manner, incubating at 37 ℃ for 1h, discarding virus liquid, flushing the cells, covering 1% agarose gel phenol-free DMEM culture medium, culturing for 2d, picking cell plaques by using an aseptic gun head to obtain purified viruses, inoculating the purified strains to the cells again for plaque purification, finally obtaining a mouse adaptive strain WBP-1 which is stable and lethal to mice, and preserving the 11 th generation mouse adaptive stable strains. The strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation at 8 months and 17 days in 2020, and is classified and named as follows: the novel coronavirus SARS-CoV-2/WBP-1 has the preservation number of CCTCC NO: v202031, address: wuhan university in Wuhan, China.

And (3) carrying out whole genome sequencing on the SARS-CoV-2/WBP-1 mouse adaptive strain obtained by purification, wherein the sequence is shown as SEQ ID NO. 1.

The novel coronavirus RT-PCR detection and identification primers are as follows:

F：5’-CCAGATGATTTTACAGGCTGCGTTA-3’

R：5’-TGTCAAGAATCTCAAGTGTCTGTGGATC-3’

the length of the amplified product fragment is 484 bp.

The preserved mouse adaptive strain WBP-1 is purified on VERO cells for 3 times, and then is continuously passaged on the VERO cells for 3 times, each generation of virus can kill mice, and the virus is not mutated by PCR and is stable in passage.

2. The purification of Igs antibodies in immune milk and non-immune milk comprises the following specific experimental steps:

(1) milk pretreatment: storage of milk at-20 ℃ milk was thawed at 4 ℃, centrifuged at 7000rpm for 30min at 4 ℃, the upper fat layer was carefully removed, the lower liquid layer was transferred to a new centrifuge tube and filtered again to remove lipids as much as possible.

(2) The new crown immune milk and non-immune milk firstly remove casein by an isoelectric point precipitation method: adding PBS with the same volume as the filtered and degreased milk, adjusting the pH value to 4.6, carrying out water bath at 40 ℃ for 30min, continuously stirring, standing overnight at 4 ℃, centrifuging at 7000rp m and 4 ℃ for 30min, removing precipitates, and transferring the supernatant to a clean beaker.

(3) Precipitating the antibody by an ammonium sulfate method: adding saturated ammonium sulfate with the same volume as that of the supernatant and having a pH of 7.4, stirring for 30min by a magnetic stirrer, standing overnight at 4 ℃, centrifuging for 30min at 7000rpm and 4 ℃, removing the supernatant, and re-dissolving the precipitate with 200mL of PBS.

(4) Dialysis desalting of reconstituted protein: centrifuging the above re-dissolved protein sample at 12000rpm at 4 deg.C for 30min to remove insoluble protein precipitate, loading the supernatant into dialysis bag for desalting, dialyzing with 10mmol of Tris-HCl (pH 9.0) for 48-72h, transferring the protein sample from the dialysis bag into a centrifuge tube, centrifuging at 12000rpm for 30min to remove protein precipitate, and transferring the supernatant into a new centrifuge tube.

(5) Protein G sepharose purification of antibodies: and (3) enabling the supernatant obtained in the step (4) to pass through a Protein G agarose gel column, washing the purified antibody after passing through the column by using precooled TBS to remove unbound and non-specifically bound proteins, eluting the antibody by using 10mL of 50mM glycine with the pH value of 2.7 after washing, and collecting eluent.

(6) Concentration of purified antibody: centrifuging and concentrating the eluate with ultrafiltration tube with molecular weight cut-off of 10kD, and packaging the concentrated sample into 1.5mL centrifuge tubes, each having a volume of 1mL, and storing at-80 deg.C

(7) Determination of purified antibody protein concentration: and (3) measuring the content of the purified and concentrated antibody protein by using a Bradford protein concentration measuring kit, and adjusting the concentrations of the immune milk and the non-immune purified sample to be consistent, wherein the content of the antibody protein is 5 mg/mL.

3. Mouse animal experiments:

(1) the 20 BALB/c mice of 6-8 weeks old were randomly divided into 4 groups of 5 mice each, one group of immune milk purified antibody protein gavage (IM-i.a), one group of immune milk purified antibody protein intraperitoneal injection (IM-i.p), and the other 2 groups of non-immune milk purified antibody protein gavage (NIM-i.a) and intraperitoneal injection (NIM-i.p), respectively. Gavage is performed once a day, and each mouse has 200 mu L; intraperitoneal injections were given once a day, 200 μ L per mouse.

(2) After continuous 5 days of gastric lavage and injection, mice were infected with the novel coronavirus SARS-CoV-2/WBP-1 virus, and all mice were infected with 5 LD by nasal drip₅₀WBP-1 New coronavirus.

(3) The mice are weighed before and after infection respectively, the weight and symptoms of the mice are observed and recorded every day, padding, drinking water and food are replaced and added at regular time, and dead mice are taken out in time for dissection until the experiment is completed.

(4) After 5 days of challenge, all the surviving mice were sacrificed, lung, trachea and turbinate bone tissues were dissected and removed, and the content of the novel coronavirus SARS-CoV-2/WBP-1 virus in the tissue samples was determined by RT-PCR.

(5) And drawing a weight change curve, a survival rate curve and a tissue virus vector graph according to the statistical result.

The experimental results are as follows:

the results of the body weight change of mice are shown in fig. 3 and table 5, and the immune milk injection group (IM-i.p) can significantly reduce the body weight loss caused by SARS-CoV-2 virus infection compared to the non-immune milk injection group (NIM-i.p); meanwhile, the weight reduction tendency of the immunized milk lavage group (IM-i.a) is lower than that of the non-immunized milk lavage group (NIM-i.a). The weight reduction tendency of the immune milk is lower than that of non-immune milk whether the milk is perfused or injected. The weight percentage of mice was calculated as mean weight of mice in each group at different times after infection with virus/mean weight of mice in each group at day 0 of infection%

TABLE 5 Effect of immune milk purified antibody protein on mouse body weight following SARS-CoV-2/WBP-1 Virus infection

The results of the mouse survival rate are shown in fig. 4 and table 6, and the mouse survival rate was calculated by counting the number of surviving mice per group/total number of infected mice per group 100%, 100% of mice in the immunized milk injection group (IM-i.p) survived, 40% of mice in the immunized milk gavage group (IM-i.a) survived, and 20% of mice in both the non-immunized milk injection group (NIM-i.p) and the gavage group (NIM-i.a).

TABLE 6 influence of immune milk purified antibody protein on survival of mice after SARS-CoV-2/WBP-1 Virus infection

As shown in FIG. 5 and Table 7, the SARS-CoV-2 viral load in different tissue samples of mice was detected, and the immune milk injection group (IM-i.p) turbinate (turbinate) could not detect the novel coronavirus SARS-CoV-2/WBP-1, and the other 3 groups could detect the novel coronavirus SARS-CoV-2/WBP-1; the content of SARS-CoV-2 virus in the immunized milk injection group (IM-i.p) trachea (trachea) and lung (lung) was significantly lower than in the other 3 groups. In the turbinate and trachea, the SARS-CoV-2 virus content in the immunized milk gavage group (I M-i.a) was also lower than that in the non-immunized milk injection group (NIM-i.p) and the gavage group (NIM-i.a).

TABLE 7 Effect of immune milk purified antibody protein on New coronavirus load in lung, trachea and turbinate tissues of mice after SARS-CoV-2/WBP-1 Virus infection (log10copies/mL)

The above results indicate that the immune milk has a significant effect against the novel coronavirus SARS-CoV-2/WBP-1 in mice.

Sequence listing

<110> university of agriculture in Huazhong

WUHAN ACADEMY OF AGRICULTURAL SCIENCES

WUHAN KEQIAN BIOLOGICAL Co.,Ltd.

<120> preparation method and application of novel coronavirus, specific antibody-containing milk or serum produced by immune dairy cow thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29218

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgagtgcact aagcatgcag ccgagtgaca gccacacaga ttttaaagtt cgtttagaga 60

acagatctac aagagatcga aagttggttg gtttgttacc tgggaaggta taaaccaacc 120

aactttcgat ctcttgtaga tctgttctct aaacgaactt taaaatctgt gtggctgtca 180

ctcggctgca tgcttagtgc actcacgcag tataattaat aactaattac tgtcgttgac 240

aggacacgag taactcgtct atcttctgca ggctgcttac ggtttcgtcc gtgttgcagc 300

cgatcatcag cacatctagg tttcgtccgg gtgtgaccga aaggtaagat ggagagcctt 360

gtccctggtt tcaacgagaa aacacacgtc caactcagtt tgcctgtttt acaggttcgc 420

gacgtgctcg tacgtggctt tggagactcc gtggaggagg tcttatcaga ggcacgtcaa 480

catcttaaag atggcacttg tggcttagta gaagttgaaa aaggcgtttt gcctcaactt 540

gaacagccct atgtgttcat caaacgttcg gatgctcgaa ctgcacctca tggtcatgtt 600

atggttgagc tggtagcaga actcgaaggc attcagtacg gtcgtagcgg tgagacactt 660

ggtgtccttg tccctcatgt gggcgaaata ccagtggctt accgcaaggt tcttcttcgt 720

aagaacggta ataaaggagc tggtggccat agttacggcg ccgatctaaa gtcatttgac 780

ttaggcgacg agcttggcac tgatccttat gaagattttc aagaaaactg gaacactaaa 840

catagcagtg gtgttacccg tgaactcatg cgtgagctta acggaggggc atacactcgc 900

tatgtcgata acaacttctg tggccctgat ggctaccctc ttgagtgcat taaagacctt 960

ctagcacgtg ctggtaaagc ttcatgcact ttgtccgaac aactggactt tattgacact 1020

aagaggggtg tatactgctg ccgtgaacat gagcatgaaa ttgcttggta cacggaacgt 1080

tctgaaaaga gctatgaatt gcagacacct tttgaaatta aattggcaaa gaaatttgac 1140

accttcaatg gggaatgtcc aaattttgta tttcccttaa attccataat caagactatt 1200

caaccaaggg ttgaaaagaa aaagcttgat ggctttatgg gtagaattcg atctgtctat 1260

ccagttgcgt caccaaatga atgcaaccaa atgtgccttt caactctcat gaagtgtgat 1320

cattgtggtg aaacttcatg gcagacgggc gattttgtta aagccacttg cgaattttgt 1380

ggcactgaga atttgactaa agaaggtgcc actacttgtg gttacttacc ccaaaatgct 1440

gttgttaaaa tttattgtcc agcatgtcac aattcagaag taggacctga gcatagtctt 1500

gccgaatacc ataatgaatc tggcttgaaa accattcttc gtaagggtgg tcgcactatt 1560

gcctttggag gctgtgtgtt ctcttatgtt ggttgccata acaagtgtgc ctattgggtt 1620

ccacgtgcta gcgctaacat aggttgtaac catacaggtg ttgttggaga aggttccgaa 1680

ggtcttaatg acaaccttct tgaaatactc caaaaagaga aagtcaacat caatattgtt 1740

ggtgacttta aacttaatga agagatcgcc attattttgg catctttttc tgcttccaca 1800

agtgcttttg tggaaactgt gaaaggtttg gattataaag cattcaaaca aattgttgaa 1860

tcctgtggta attttaaagt tacaaaagga aaagctaaaa aaggtgcctg gaatattggt 1920

gaacagaaat caatactgag tcctctttat gcatttgcat cagaggctgc tcgtgttgta 1980

cgatcaattt tctctcgcac tcttgaaact gctcaaaatt ctgtgcgtgt tttacagaag 2040

gccgctataa caatactaga tggaatttca cagtattcac tgagactcat tgatgctatg 2100

atgttcacat ctgatttggc tactaacaat ctagttgtaa tggcctacat tacaggtggt 2160

gttgttcagt tgacttcgca gtggctaact aacatctttg gcactgttta tgaaaaactc 2220

aaacccgtcc ttgattggct tgaagagaag tttaaggaag gtgtagagtt tcttagagac 2280

ggttgggaaa ttgttaaatt tatctcaacc tgtgcttgtg aaattgtcgg tggacaaatt 2340

gtcacctgtg caaaggaaat taaggagagt gttcagacat tctttaagct tgtaaataaa 2400

tttttggctt tgtgtgctga ctctatcatt attggtggag ctaaacttaa agccttgaat 2460

ttaggtgaaa catttgtcac gcactcaaag ggattgtaca gaaagtgtgt taaatccaga 2520

gaagaaactg gcctactcat gcctctaaaa gccccaaaag aaattatctt cttagaggga 2580

gaaacacttc ccacagaagt gttaacagag gaagttgtct tgaaaactgg tgatttacaa 2640

ccattagaac aacctactag tgaagctgtt gaagctccat tggttggtac accagtttgt 2700

attaacgggc ttatgttgct cgaaatcaaa gacacagaaa agtactgtgc ccttgcacct 2760

aatatgatgg taacaaacaa taccttcaca ctcaaaggcg gtgcaccaac aaaggttact 2820

tttggtgatg acactgtgat agaagtgcaa ggttacaaga gtgtgaatat cacttttgaa 2880

cttgatgaaa ggattgataa agtacttaat gagaagtgct ctgcctatac agttgaactc 2940

ggtacagaag taaatgagtt cgcctgtgtt gtggcagatg ctgtcataaa aactttgcaa 3000

ccagtatctg aattacttac accactgggc attgatttag atgagtggag tatggctaca 3060

tactacttat ttgatgagtc tggtgagttt aaattggctt cacatatgta ttgttctttc 3120

taccctccag atgaggatga agaagaaggt gattgtgaag aagaagagtt tgagccatca 3180

actcaatatg agtatggtac tgaagatgat taccaaggta aacctttgga atttggtgcc 3240

acttctgctg ctcttcaacc tgaagaagag caagaagaag attggttaga tgatgatagt 3300

caacaaactg ttggtcaaca agacggcagt gaggacaatc agacaactac tattcaaaca 3360

attgttgagg ttcaacctca attagagatg gaacttacac cagttgttca gactattgaa 3420

gtgaatagtt ttagtggtta tttaaaactt actgacaatg tatacattaa aaatgcagac 3480

attgtggaag aagctaaaaa ggtaaaacca acagtggttg ttaatgcagc caatgtttac 3540

cttaaacatg gaggaggtgt tgcaggagcc ttaaataagg ctactaacaa tgccatgcaa 3600

gttgaatctg atgattacat agctactaat ggaccactta aagtgggtgg tagttgtgtt 3660

ttaagcggac acaatcttgc taaacactgt cttcatgttg tcggcccaaa tgttaacaaa 3720

ggtgaagaca ttcaacttct taagagtgct tatgaaaatt ttaatcagca cgaagttcta 3780

cttgcaccat tattatcagc tggtattttt ggtgctgacc ctatacattc tttaagagtt 3840

tgtgtagata ctgttcgcac aaatgtctac ttagctgtct ttgataaaaa tctctatgac 3900

aaacttgttt caagcttttt ggaaatgaag agtgaaaagc aagttgaaca aaagatcgct 3960

gagattccta aagaggaagt taagccattt ataactgaaa gtaaaccttc agttgaacag 4020

agaaaacaag atgataagaa aatcaaagct tgtgttgaag aagttacaac aactctggaa 4080

gaaactaagt tcctcacaga aaacttgtta ctttatattg acattaatgg caatcttcat 4140

ccagattctg ccactcttgt tagtgacatt gacatcactt tcttaaagaa agatgctcca 4200

tatatagtgg gtgatgttgt tcaagagggt gttttaactg ctgtggttat acctactaaa 4260

aaggctggtg gcactactga aatgctagcg aaagctttga gaaaagtgcc aacagacaat 4320

tatataacca cttacccggg tcagggttta aatggttaca ctgtagagga ggcaaagaca 4380

gtgcttaaaa agtgtaaaag tgccttttac attctaccat ctattatctc taatgagaag 4440

caagaaattc ttggaactgt ttcttggaat ttgcgagaaa tgcttgcaca tgcagaagaa 4500

acacgcaaat taatgcctgt ctgtgtggaa actaaagcca tagtttcaac tatacagcgt 4560

aaatataagg gtattaaaat acaagagggt gtggttgatt atggtgctag attttacttt 4620

tacaccagta aaacaactgt agcgtcactt atcaacacac ttaacgatct aaatgaaact 4680

cttgttacaa tgccacttgg ctatgtaaca catggcttaa atttggaaga agctgctcgg 4740

tatatgagat ctctcaaagt gccagctaca gtttctgttt cttcacctga tgctgttaca 4800

gcgtataatg gttatcttac ttcttcttct aaaacacctg aagaacattt tattgaaacc 4860

atctcacttg ctggttccta taaagattgg tcctattctg gacaatctac acaactaggt 4920

atagaatttc ttaagagagg tgataaaagt gtatattaca ctagtaatcc taccacattc 4980

cacctagatg gtgaagttat cacctttgac aatcttaaga cacttctttc tttgagagaa 5040

gtgaggacta ttaaggtgtt tacaacagta gacaacatta acctccacac gcaagttgtg 5100

gacatgtcaa tgacatatgg acaacagttt ggtccaactt atttggatgg agctgatgtt 5160

actaaaataa aacctcataa ttcacatgaa ggtaaaacat tttatgtttt acctaatgat 5220

gacactctac gtgttgaggc ttttgagtac taccacacaa ctgatcctag ttttctgggt 5280

aggtacatgt cagcattaaa tcacactaaa aagtggaaat acccacaagt taatggttta 5340

acttctatta aatgggcaga taacaactgt tatcttgcca ctgcattgtt aacactccaa 5400

caaatagagt tgaagtttaa tccacctgct ctacaagatg cttattacag agcaagggct 5460

ggtgaagctg ctaacttttg tgcacttatc ttagcctact gtaataagac agtaggtgag 5520

ttaggtgatg ttagagaaac aatgagttac ttgtttcaac atgccaattt agattcttgc 5580

aaaagagtct tgaacgtggt gtgtaaaact tgtggacaac agcagacaac ccttaagggt 5640

gtagaagctg ttatgtacat gggcacactt tcttatgaac aatttaagaa aggtgttcag 5700

ataccttgta cgtgtggtaa acaagctaca aaatatctag tacaacagga gtcacctttt 5760

gttatgatgt cagcaccacc tgctcagtat gaacttaagc atggtacatt tacttgtgct 5820

agtgagtaca ctggtaatta ccagtgtggt cactataaac atataacttc taaagaaact 5880

ttgtattgca tagacggtgc tttacttaca aagtcctcag aatacaaagg tcctattacg 5940

gatgttttct acaaagaaaa cagttacaca acaaccataa aaccagttac ttataaattg 6000

gatggtgttg tttgtacaga aattgaccct aagttggaca attattataa gaaagacaat 6060

tcttatttca cagagcaacc aattgatctt gtaccaaacc aaccatatcc aaacgcaagc 6120

ttcgataatt ttaagtttgt atgtgataat atcaaatttg ctgatgattt aaaccagtta 6180

actggttata agaaacctgc ttcaagagag cttaaagtta catttttccc tgacttaaat 6240

ggtgatgtgg tggctattga ttataaacac tacacaccct cttttaagaa aggagctaaa 6300

ttgttacata aacctattgt ttggcatgtt aacaatgcaa ctaataaagc cacgtataaa 6360

ccaaatacct ggtgtatacg ttgtctttgg agcacaaaac cagttgaaac atcaaattcg 6420

tttgatgtac tgaagtcaga ggacgcgcag ggaatggata atcttgcctg cgaagatcta 6480

aaaccagtct ctgaagaagt agtggaaaat cctaccatac agaaagacgt tcttgagtgt 6540

aatgtgaaaa ctaccgaagt tgtaggagac attatactta aaccagcaaa taatagttta 6600

aaaattacag aagaggttgg ccacacagat ctaatggctg cttatgtaga caattctagt 6660

cttactatta agaaacctaa tgaattatct agagtattag gtttgaaaac ccttgctact 6720

catggtttag ctgctgttaa tagtgtccct tgggatacta tagctaatta tgctaagcct 6780

tttcttaaca aagttgttag tacaactact aacatagtta cacggtgttt aaaccgtgtt 6840

tgtactaatt atatgcctta tttctttact ttattgctac aattgtgtac ttttactaga 6900

agtacaaatt ctagaattaa agcatctatg ccgactacta tagcaaagaa tactgttaag 6960

agtgtcggta aattttgtct agaggcttca tttaattatt tgaagtcacc taatttttct 7020

aaactgataa atattataat ttggttttta ctattaagtg tttgcctagg ttctttaatc 7080

tactcaaccg ctgctttagg tgttttaatg tctaatttag gcatgccttc ttactgtact 7140

ggttacagag aaggctattt gaactctact aatgtcacta ttgcaaccta ctgtactggt 7200

tctatacctt gtagtgtttg tcttagtggt ttagattctt tagacaccta tccttcttta 7260

gaaactatac aaattaccat ttcatctttt aaatgggatt taactgcttt tggcttagtt 7320

gcagagtggt ttttggcata tattcttttc actaggtttt tctatgtact tggattggct 7380

gcaatcatgc aattgttttt cagctatttt gcagtacatt ttattagtaa ttcttggctt 7440

atgtggttaa taattaatct tgtacaaatg gccccgattt cagctatggt tagaatgtac 7500

atcttctttg catcatttta ttatgtatgg aaaagttatg tgcatgttgt agacggttgt 7560

aattcatcaa cttgtatgat gtgttacaaa cgtaatagag caacaagagt cgaatgtaca 7620

actattgtta atggtgttag aaggtccttt tatgtctatg ctaatggagg taaaggcttt 7680

tgcaaactac acaattggaa ttgtgttaat tgtgatacat tctgtgctgg tagtacattt 7740

attagtgatg aagttgcgag agacttgtca ctacagttta aaagaccaat aaatcctact 7800

gaccagtctt cttacatcgt tgatagtgtt acagtgaaga atggttccat ccatctttac 7860

tttgataaag ctggtcaaaa gacttatgaa agacattctc tctctcattt tgttaactta 7920

gacaacctga gagctaataa cactaaaggt tcattgccta ttaatgttat agtttttgat 7980

ggtaaatcaa aatgtgaaga atcatctgca aaatcagcgt ctgtttacta cagtcagctt 8040

atgtgtcaac ctatactgtt actagatcag gcattagtgt ctgatgttgg tgatagtgcg 8100

gaagttgcag ttaaaatgtt tgatgcttac gttaatacgt tttcatcaac ttttaacgta 8160

ccaatggaaa aactcaaaac actagttgca actgcagaag ctgaacttgc aaagaatgtg 8220

tccttagaca atgtcttatc tacttttatt tcagcagctc ggcaagggtt tgttgattca 8280

gatgtggaaa ctaaagatgt tgttgaatgt cttaaattgt cacatcaatc tgacatagaa 8340

gttactggcg atagttgtaa taactatatg ctcacctata acaaagttga aaacatgaca 8400

ccccgtgacc ttggtgcttg tattgactgt agtgcgcgtc atattaatgc gcaggtagca 8460

aaaagtcaca acattgcttt gatatggaac gttaaagatt tcatgtcatt gtctgaacaa 8520

ctacgaaaac aaatacgtag tgctgctaaa aagaataact taccttttaa gttgacatgt 8580

gcaactacta gacaagttgt taatgttgta acaacaaaga tagcacttaa gggtggtaaa 8640

attgttaata attggttgaa gcagttaatt aaagttacac ttgtgttcct ttttgttgct 8700

gctattttct atttaataac acctgttcat gtcatgtcta aacatactga cttttcaagt 8760

gaaatcatag gatacaaggc tattgatggt ggtgtcactc gtgacatagc atctacagat 8820

acttgttttg ctaacaaaca tgctgatttt gacacatggt ttagccagcg tggtggtagt 8880

tatactaatg acaaagcttg cccattgatt gctgcagtca taacaagaga agtgggtttt 8940

gtcgtgcctg gtttgcctgg cacgatatta cgcacaacta atggtgactt tttgcatttc 9000

ttacctagag tttttagtgc agttggtaac atctgttaca caccatcaaa acttatagag 9060

tacactgact ttgcaacatc agcttgtgtt ttggctgctg aatgtacaat ttttaaagat 9120

gcttctggta agccagtacc atattgttat gataccaatg tactagaagg ttctgttgct 9180

tatgaaagtt tacgccctga cacacgttat gtgctcatgg atggctctat tattcaattt 9240

cctaacacct accttgaagg ttctgttaga gtggtaacaa cttttgattc tgagtactgt 9300

aggcacggca cttgtgaaag atcagaagct ggtgtttgtg tatctactag tggtagatgg 9360

gtacttaaca atgattatta cagatcttta ccaggagttt tctgtggtgt agatgctgta 9420

aatttactta ctaatatgtt tacaccacta attcaaccta ttggtgcttt ggacatatca 9480

gcatctatag tagctggtgg tattgtagct atcgtagtaa catgccttgc ctactatttt 9540

atgaggttta gaagagcttt tggtgaatac agtcatgtag ttgcctttaa tactttacta 9600

ttccttatgt cattcactgt actctgttta acaccagttt actcattctt acctggtgtt 9660

tattctgtta tttacttgta cttgacattt tatcttacta atgatgtttc ttttttagca 9720

catattcagt ggatggttat gttcacacct ttagtacctt tctggataac aattgcttat 9780

atcatttgta tttccacaaa gcatttctat tggttcttta gtaattacct aaagagacgt 9840

gtagtcttta atggtgtttc ctttagtact tttgaagaag ctgcgctgtg cacctttttg 9900

ttaaataaag aaatgtatct aaagttgcgt agtgatgtgc tattacctct tacgcaatat 9960

aatagatact tagctcttta taataagtac aagtatttta gtggagcaat ggatacaact 10020

agctacagag aagctgcttg ttgtcatctc gcaaaggctc tcaatgactt cagtaactca 10080

ggttctgatg ttctttacca accaccacaa acctctatca cctcagctgt tttgcagagt 10140

ggttttagaa aaatggcatt cccatctggt aaagttgagg gttgtatggt acaagtaact 10200

tgtggtacaa ctacacttaa cggtctttgg cttgatgacg tagtttactg tccaagacat 10260

gtgatctgca cctctgaaga catgcttaac cctaattatg aagatttact cattcgtaag 10320

tctaatcata atttcttggt acaggctggt aatgttcaac tcagggttat tggacattct 10380

atgcaaaatt gtgtacttaa gcttaaggtt gatacagcca atcctaagac acctaagtat 10440

aagtttgttc gcattcaacc aggacagact ttttcagtgt tagcttgtta caatggttca 10500

ccatctggtg tttaccaatg tgctatgagg cccaatttca ctattaaggg ttcattcctt 10560

aatggttcat gtggtagtgt tggttttaac atagattatg actgtgtctc tttttgttac 10620

atgcaccata tggaattacc aactggagtt catgctggca cagacttaga aggtaacttt 10680

tatggacctt ttgttgacag gcaaacagca caagcagctg gtacggacac aactattaca 10740

gttaatgttt tagcttggtt gtacgctgct gttataaatg gagacaggtg gtttctcaat 10800

cgatttacca caactcttaa tgactttaac cttgtggcta tgaagtacaa ttatgaacct 10860

ctaacacaag accatgttga catactagga cctctttctg ctcaaactgg aattgccgtt 10920

ttagatatgt gtgcttcatt aaaagaatta ctgcaaaatg gtatgaatgg acgtaccata 10980

ttgggtagtg ctttattaga agatgaattt acacctcttg atgttgttag acaatgctca 11040

ggtgttactt tccaaagtgc agtgaaaaga acaaccaagg gtacacacca ctggttgtta 11100

ctcacaattt tgacttcact tttagtttta gtccagagta ctcaatggtc tttgttcttt 11160

tttttgtatg aaaatgcctt tttacctttt gctatgggta ttattgctat gtctgctttt 11220

gcaatgatgt ttgtcaaaca taagcatgca tttctctgtt tgtttttgtt accttctctt 11280

gccactgtag cttattttaa tatggtctat atgcctgcta gttgggtgat gcgtattatg 11340

acatggttgg atatggttga tactagtttg tctggtttta agctaaaaga ctgtgttatg 11400

tatgcatcag ctgtagtgtt actaatcctt atgacagcaa gaactgtgta tgatgatggt 11460

gctaggagag tgtggacact tatgaatgtc ttgacactcg tttataaagt ttattatggt 11520

aatgctttag atcaagccat ttccatgtgg gctcttataa tctctgttac ttctaactac 11580

tcaggtgtag ttacaactgt catgtttttg gccagaggta ttgtttttat gtgtgttgag 11640

tattgcccta ttttcttcat aactggtaat acacttcagt gtataatgct agtttattgt 11700

ttcttaggct atttttgtac ttgttacttt ggcctctttt gtttactcaa ccgctacttt 11760

agactgactc ttggtgttta tgattactta gtttctacac aggagtttag atatatgaat 11820

tcacagggac tactcccacc caagaatagc atagatgcct tcaaactcaa cattaaattg 11880

ttgggtgttg gtggcaaacc ttgtatcaaa gtagccactg tacagtctaa aatgtcagat 11940

gtaaagtgca catcagtagt cttactctca gttttgcaac aactcagagt agaatcatca 12000

tctaaattgt gggctcaatg tgtccagtta cacaatgaca ttctcttagc taaagatact 12060

actgaagcct ttgaaaaaat ggtttcacta ctttctgttt tgctttccat gcagggtgct 12120

gtagacataa acaagctttg tgaagaaatg ctggacaaca gggcaacctt acaagctata 12180

gcctcagagt ttagttccct tccatcatat gcagcttttg ctactgctca agaagcttat 12240

gagcaggctg ttgctaatgg tgattctgaa gttgttctta aaaagttgaa gaagtctttg 12300

aatgtggcta aatctgaatt tgaccgtgat gcagccatgc aacgtaagtt ggaaaagatg 12360

gctgatcaag ctatgaccca aatgtataaa caggctagat ctgaggacaa gagggcaaaa 12420

gttactagtg ctatgcagac aatgcttttc actatgctta gaaagttgga taatgatgca 12480

ctcaacaaca ttatcaacaa tgcaagagat ggttgtgttc ccttgaacat aatacctctt 12540

acaacagcag ccaaactaat ggttgtcata ccagactata acacatataa aaatacgtgt 12600

gatggtacaa catttactta tgcatcagca ttgtgggaaa tccaacaggt tgtagatgca 12660

gatagtaaaa ttgttcaact tagtgaaatt agtatggaca attcacctaa tttagcatgg 12720

cctcttattg taacagcttt aagggccaat tctgctgtca aattacagaa taatgagctt 12780

agtcctgttg cactacgaca gatgtcttgt gctgccggta ctacacaaac tgcttgcact 12840

gatgacaatg cgttagctta ctacaacaca acaaagggag gtaggtttgt acttgcactg 12900

ttatccgatt tacaggattt gaaatgggct agattcccta agagtgatgg aactggtact 12960

atctatacag aactggaacc accttgtagg tttgttatag acacacctaa aggtcctaaa 13020

gtgaagtatt tatactttat taaaggatta aacaacctaa atagaggtat ggtacttggt 13080

agtttagctg ccacagtacg tctacaagct ggtaatgcaa cagaagtgcc tgccaattca 13140

actgtattat ctttctgtgc ttttgctgta gatgctgcta aagcttacaa agattatcta 13200

gctagtgggg gacaaccaat cactaattgt gttaagatgt tgtgtacaca cactggtact 13260

ggtcaggcaa taacagttac accggaagcc aatatggatc aagaatcctt tggtggtgca 13320

tcgtgttgtc tgtactgccg ttgccacata gatcatccaa atcctaaagg attttgtgac 13380

ttaaaaggta agtatgtaca aatacctaca acttgtgcta atgaccctgt gggttttaca 13440

cttaaaaaca cagtctgtac cgtctgcggt atgtggaaag gttatggctg tagttgtgat 13500

caactccgcg aacccatgct tcagtcagct gatgcacaat cgtttttaaa cgggtttgcg 13560

gtgtaagtgc agcccgtctt acaccgtgcg gcacaggcac tagtactgat gtcgtataca 13620

gggcttttga catctacaat gataaagtag ctggttttgc taaattccta aaaactaatt 13680

gttgtcgctt ccaagaaaag gacgaagatg acaatttaat tgattcttac tttgtagtta 13740

agagacacac tttctctaac taccaacatg aagaaacaat ttataattta cttaaggatt 13800

gtccagctgt tgctaaacat gacttcttta agtttagaat agacggtgac atggtaccac 13860

atatatcacg tcaacgtctt actaaataca caatggcaga cctcgtctat gctttaaggc 13920

attttgatga aggtaattgt gacacattaa aagaaatact tgtcacatac aattgttgtg 13980

atgatgatta tttcaataaa aaggactggt atgattttgt agaaaaccca gatatattac 14040

gcgtatacgc caacttaggt gaacgtgtac gccaagcttt gttaaaaaca gtacaattct 14100

gtgatgccat gcgaaatgct ggtattgttg gtgtactgac attagataat caagatctca 14160

atggtaactg gtatgatttc ggtgatttca tacaaaccac gccaggtagt ggagttcctg 14220

ttgtagattc ttattattca ttgttaatgc ctatattaac cttgaccagg gctttaactg 14280

cagagtcaca tgttgacact gacttaacaa agccttacat taagtgggat ttgttaaaat 14340

atgacttcac ggaagagagg ttaaaactct ttgaccgtta ttttaaatat tgggatcaga 14400

cataccaccc aaattgtgtt aactgtttgg atgacagatg cattctgcat tgtgcaaact 14460

ttaatgtttt attctctaca gtgttcccac ctacaagttt tggaccacta gtgagaaaaa 14520

tatttgttga tggtgttcca tttgtagttt caactggata ccacttcaga gagctaggtg 14580

ttgtacataa tcaggatgta aacttacata gctctagact tagttttaag gaattacttg 14640

tgtatgctgc tgaccctgct atgcacgctg cttctggtaa tctattacta gataaacgca 14700

ctacgtgctt ttcagtagct gcacttacta acaatgttgc ttttcaaact gtcaaacccg 14760

gtaattttaa caaagacttc tatgactttg ctgtgtctaa gggtttcttt aaggaaggaa 14820

gttctgttga attaaaacac ttcttctttg ctcaggatgg taatgctgct atcagcgatt 14880

atgactacta tcgttataat ctaccaacaa tgtgtgatat cagacaacta ctatttgtag 14940

ttgaagttgt tgataagtac tttgattgtt acgatggtgg ctgtattaat gctaaccaag 15000

tcatcgtcaa caacctagac aaatcagctg gttttccatt taataaatgg ggtaaggcta 15060

gactttatta tgattcaatg agttatgagg atcaagatgc acttttcgca tatacaaaac 15120

gtaatgtcat ccctactata actcaaatga atcttaagta tgccattagt gcaaagaata 15180

gagctcgcac cgtagctggt gtctctatct gtagtactat gaccaataga cagtttcatc 15240

aaaaattatt gaaatcaata gccgccacta gaggagctac tgtagtaatt ggaacaagca 15300

aattctatgg tggttggcac aacatgttaa aaactgttta tagtgatgta gaaaaccctc 15360

accttatggg ttgggattat cctaaatgtg atagagccat gcctaacatg cttagaatta 15420

tggcctcact tgttcttgct cgcaaacata caacgtgttg tagcttgtca caccgtttct 15480

atagattagc taatgagtgt gctcaagtat tgagtgaaat ggtcatgtgt ggcggttcac 15540

tatatgttaa accaggtgga acctcatcag gagatgccac aactgcttat gctaatagtg 15600

tttttaacat ttgtcaagct gtcacggcca atgttaatgc acttttatct actgatggta 15660

acaaaattgc cgataagtat gtccgcaatt tacaacacag actttatgag tgtctctata 15720

gaaatagaga tgttgacaca gactttgtga atgagtttta cgcatatttg cgtaaacatt 15780

tctcaatgat gatactctct gacgatgctg ttgtgtgttt caatagcact tatgcatctc 15840

aaggtctagt ggctagcata aagaacttta agtcagttct ttattatcaa aacaatgttt 15900

ttatgtctga agcaaaatgt tggactgaga ctgaccttac taaaggacct catgaatttt 15960

gctctcaaca tacaatgcta gttaaacagg gtgatgatta tgtgtacctt ccttacccag 16020

atccatcaag aatcctaggg gccggctgtt ttgtagatga tatcgtaaaa acagatggta 16080

cacttatgat tgaacggttc gtgtctttag ctatagatgc ttacccactt actaaacatc 16140

ctaatcagga gtatgctgat gtctttcatt tgtacttaca atacataaga aagctacatg 16200

atgagttaac aggacacatg ttagacatgt attctgttat gcttactaat gataacactt 16260

caaggtattg ggaacctgag ttttatgagg ctatgtacac accgcataca gtcttacagg 16320

ctgttggggc ttgtgttctt tgcaattcac agacttcatt aagatgtggt gcttgcatac 16380

gtagaccatt cttatgttgt aaatgctgtt acgaccatgt catatcaaca tcacataaat 16440

tagtcttgtc tgttaatccg tatgtttgca atgctccagg ttgtgatgtc acagatgtga 16500

ctcaacttta cttaggaggt atgagctatt attgtaaatc acataaacca cccattagtt 16560

ttccattgtg tgctaatgga caagtttttg gtttatataa aaatacatgt gttggtagcg 16620

ataatgttac tgactttaat gcaattgcaa catgtgactg gacaaatgct ggtgattaca 16680

ttttagctaa cacctgtact gaaagactca agctttttgc agcagaaacg ctcaaagcta 16740

ctgaggagac atttaaactg tcttatggta ttgctactgt acgtgaagtg ctgtctgaca 16800

gagaattaca tctttcatgg gaagttggta aacctagacc accacttaac cgaaattatg 16860

tctttactgg ttatcgtgta actaaaaaca gtaaagtaca aataggagag tacacctttg 16920

aaaaaggtga ctatggtgat gctgttgttt accgaggtac aacaacttac aaattaaatg 16980

ttggtgatta ttttgtgctg acatcacata cagtaatgcc attaagtgca cctacactag 17040

tgccacaaga gcactatgtt agaattactg gcttataccc aacactcaat atctcagatg 17100

agttttctag caatgttgca aattatcaaa aggttggtat gcaaaagtat tctacactcc 17160

agggaccacc tggtactggt aagagtcatt ttgctattgg cctagctctc tactaccctt 17220

ctgctcgcat agtgtataca gcttgctctc atgccgctgt tgatgcacta tgtgagaagg 17280

cattaaaata tttgcctata gataaatgta gtagaattat acctgcacgt gctcgtgtag 17340

agtgttttga taaattcaaa gtgaattcaa cattagaaca gtatgtcttt tgtactgtaa 17400

atgcattgcc tgagacgaca gcagatatag ttgtctttga tgaaatttca atggccacaa 17460

attatgattt gagtgttgtc aatgccagat tacgtgctaa gcactatgtg tacattggcg 17520

accctgctca attacctgca ccacgcacat tgctaactaa gggcacacta gaaccagaat 17580

atttcaattc agtgtgtaga cttatgaaaa ctataggtcc agacatgttc ctcggaactt 17640

gtcggcgttg tcctgctgaa attgttgaca ctgtgagtgc tttggtttat gataataagc 17700

ttaaagcaca taaagacaaa tcagctcaat gctttaaaat gttttataag ggtgttatca 17760

cgcatgatgt ttcatctgca attaacaggc cacaaatagg cgtggtaaga gaattcctta 17820

cacgtaaccc tgcttggaga aaagctgtct ttatttcacc ttataattca cagaatgctg 17880

tagcctcaaa gattttggga ctaccaactc aaactgttga ttcatcacag ggctcagaat 17940

atgactatgt catattcact caaaccactg aaacagctca ctcttgtaat gtaaacagat 18000

ttaatgttgc tattaccaga gcaaaagtag gcatactttg cataatgtct gatagagacc 18060

tttatgacaa gttgcaattt acaagtcttg aaattccacg taggaatgtg gcaactttac 18120

aagctgaaaa tgtaacagga ctctttaaag attgtagtaa ggtaatcact gggttacatc 18180

ctacacaggc acctacacac ctcagtgttg acactaaatt caaaactgaa ggtttatgtg 18240

ttgacatacc tggcatacct aaggacatga cctatagaag actcatctct atgatgggtt 18300

ttaaaatgaa ttatcaagtt aatggttacc ctaacatgtt tatcacccgc gaagaagcta 18360

taagacatgt acgtgcatgg attggcttcg atgtcgaggg gtgtcatgct actagagaag 18420

ctgttggtac caatttacct ttacagctag gtttttctac aggtgttaac ctagttgctg 18480

tacctacagg ttatgttgat acacctaata atacagattt ttccagagtt agtgctaaac 18540

caccgcctgg agatcaattt aaacacctca taccacttat gtacaaagga cttccttgga 18600

atgtagtgcg tataaagatt gtacaaatgt taagtgacac acttaaaaat ctctctgaca 18660

gagtcgtatt tgtcttatgg gcacatggct ttgagttgac atctatgaag tattttgtga 18720

aaataggacc tgagcgcacc tgttgtctat gtgatagacg tgccacatgc ttttccactg 18780

cttcagacac ttatgcctgt tggcatcatt ctattggatt tgattacgtc tataatccgt 18840

ttatgattga tgttcaacaa tggggtttta caggtaacct acaaagcaac catgatctgt 18900

attgtcaagt ccatggtaat gcacatgtag ctagttgtga tgcaatcatg actaggtgtc 18960

tagctgtcca cgagtgcttt gttaagcgtg ttgactggac tattgaatat cctataattg 19020

gtgatgaact gaagattaat gcggcttgta gaaaggttca acacatggtt gttaaagctg 19080

cattattagc agacaaattc ccagttcttc acgacattgg taaccctaaa gctattaagt 19140

gtgtacctca agctgatgta gaatggaagt tctatgatgc acatccttgt agtgacaaag 19200

cttataaaat agaagaatta ttctattctt atgccacaca ttctgacaaa ttcacagatg 19260

gtgtatgcct attttggaat tgcaatgtcg atagatatcc tgctaattcc attgtttgta 19320

gatttgacac tagagtgcta tctaacctta acttgcctgg ttgtgatggt ggcagtttgt 19380

atgtaaataa acatgcattc cacacaccag cttttgataa aagtgctttt gttaatttaa 19440

aacaattacc atttttctat tactctgaca gtccatgtga gtctcatgga aaacaagtag 19500

tgtcagatat agattatgta ccactaaagt ctgctacgtg tataacacgt tgcaatttag 19560

gtggtgctgt ctgtagacat catgctaatg agtacagatt gtatctcgat gcttataaca 19620

tgatgatctc agctggcttt agcttgtggg tttacaaaca atttgatact tataacctct 19680

ggaacacttt tacaagactt cagagtttag aaaatgtggc ttttaatgtt gtaaataagg 19740

gacactttga tggacaacag ggtgaagtac cagtttctat cattaataac actgtttaca 19800

caaaagttga tggtgttgat gtagaattgt ttgaaaataa aacaacatta cctgttaatg 19860

tagcatttga gctttgggct aagcgcaaca ttaaaccagt accagaggtg aaaatactca 19920

ataatttggg tgtggacatt gctgctaata ctgtgatctg ggactacaaa agagatgctc 19980

cagcacatat atctactatt ggtgtttgtt ctatgactga catagccaag aaaccaactg 20040

aaacgatttg tgcaccactc actgtctttt ttgatggtag agttgatggt caagtagact 20100

tatttagaaa tgcccgtaat ggtgttctta ttacagaagg tagtgttaaa ggtttacaac 20160

catctgtagg tcccaaacaa gctagtctta atggagtcac attaattgga gaagccgtaa 20220

aaacacagtt caattattat aagaaagttg atggtgttgt ccaacaatta cctgaaactt 20280

actttactca gagtagaaat ttacaagaat ttaaacccag gagtcaaatg gaaattgatt 20340

tcttagaatt agctatggat gaattcattg aacggtataa attagaaggc tatgccttcg 20400

aacatatcgt ttatggagat tttagtcata gtcagttagg tggtttacat ctactgattg 20460

gactagctaa acgttttaag gaatcacctt ttgaattaga agattttatt cctatggaca 20520

gtacagttaa aaactatttc ataacagatg cgcaaacagg ttcatctaag tgtgtgtgtt 20580

ctgttattga tttattactt gatgattttg ttgaaataat aaaatcccaa gatttatctg 20640

tagtttctaa ggttgtcaaa gtgactattg actatacaga aatttcattt atgctttggt 20700

gtaaagatgg ccatgtagaa acattttacc caaaattaca atctagtcaa gcgtggcaac 20760

cgggtgttgc tatgcctaat ctttacaaaa tgcaaagaat gctattagaa aagtgtgacc 20820

ttcaaaatta tggtgatagt gcaacattac ctaaaggcat aatgatgaat gtcgcaaaat 20880

atactcaact gtgtcaatat ttaaacacat taacattagc tgtaccctat aatatgagag 20940

ttatacattt tggtgctggt tctgataaag gagttgcacc aggtacagct gttttaagac 21000

agtggttgcc tacgggtacg ctgcttgtcg attcagatct taatgacttt gtctctgatg 21060

cagattcaac tttgattggt gattgtgcaa ctgtacatac agctaataaa tgggatctca 21120

ttattagtga tatgtacgac cctaagacta aaaatgttac aaaagaaaat gactctaaag 21180

agggtttttt cacttacatt tgtgggttta tacaacaaaa gctagctctt ggaggttccg 21240

tggctataaa gataacagaa cattcttgga atgctgatct ttataagctc atgggacact 21300

tcgcatggtg gacagccttt gttactaatg tgaatgcgtc atcatctgaa gcatttttaa 21360

ttggatgtaa ttatcttggc aaaccacgcg aacaaataga tggttatgtc atgcatgcaa 21420

attacatatt ttggaggaat acaaatccaa ttcagttgtc ttcctattct ttatttgaca 21480

tgagtaaatt tccccttaaa ttaaggggta ctgctgttat gtctttaaaa gaaggtcaaa 21540

tcaatgatat gattttatct cttcttagta aaggtagact tataattaga gaaaacaaca 21600

gagttgttat ttctagtgat gttcttgtta acaactaaac gaacaatgtt tgtttttctt 21660

gttttattgc cactagtctc tagtcagtgt gttaatctta caaccagaac tcaattaccc 21720

cctgcataca ctaattcttt cacacgtggt gtttattacc ctgacaaagt tttcagatcc 21780

tcagttttac attcaactca ggacttgttc ttacctttct tttctaatgt tacttggttc 21840

catgctatac atgtctctgg gaccaaaggt actaagaggt ttgataaccc tgtcctacca 21900

tttaatgatg gtgtttattt tgcttccact gagaagtcta acataataag aggctggatt 21960

tttggtacta ctttagattc gaagacccag tccctactta ttgttaataa cgctactaat 22020

gttgttatta aagtctgtga atttcaattt tgtaatgatc catttttggg tgtttattac 22080

cacaaaaaca acaaaagttg gatggaaagt gagttcagag tttattctag tgcgaataat 22140

tgcacttttg aatatgtctc tcagcctttt cttatggacc ttgaaggaaa acagggtaat 22200

ttcaaaaatc ttagggaatt tgtgtttaag aatattgatg gttattttaa aatatattct 22260

aagcacacgc ctattaattt agtgcgtgat ctccctcagg gtttttcggc tttagaacca 22320

ttggtagatt tgccaatagg tattaacatc actaggtttc aaactttact tgctttacat 22380

agaagttatt tgactcctgg tgattcttct tcaggttgga cagctggtgc tgcagcttat 22440

tatgtgggtt atcttcaacc taggactttt ctattaaaat ataatgaaaa tggaaccatt 22500

acagatgctg tagactgtgc acttgaccct ctctcagaaa caaagtgtac gttgaaatcc 22560

ttcactgtag aaaaaggaat ctatcaaact tctaacttta gagtccaacc aacagaatct 22620

attgttagat ttcctaatat tacaaacttg tgcccttttg gtgaagtttt taacgccacc 22680

agatttgcat ctgtttatgc ttggaacagg aagagaatca gcaactgtgt tgctgattat 22740

tctgtcctat ataattccgc atcattttcc acttttaagt gttatggagt gtctcctact 22800

aaattaaatg atctctgctt tactaatgtc tatgcagatt catttgtaat tagaggtgat 22860

gaagtcagac aaatcgctcc agggcaaact ggaaagattg ctgattataa ttataaatta 22920

ccagatgatt ttacaggctg cgttatagct tggaattcta acaatcttga ttctaaggtt 22980

ggtggtaatt ataattacct gtatagattg tttaggaagt ctaatctcaa accttttgag 23040

agagatattt caactgaaat ctatcaggcc ggtagcacac cttgtaatgg tgttgaaggt 23100

tttaattgtt actttccttt aaaatcatat ggtttccacc ccactaatgg tgttggttac 23160

caaccataca gagtagtagt actttctttt gaacttctac atgcaccagc aactgtttgt 23220

ggacctaaaa agtctactaa tttggttaaa aacaaatgtg tcaatttcaa cttcaatggt 23280

ttaacaggca caggtgttct tactgagtct aacaaaaagt ttctgccttt ccaacaattt 23340

ggcagagaca ttgctgacac tactgatgct gtccgtgatc cacagacact tgagattctt 23400

gacattacac catgttcttt tggtggtgtc agtgttataa caccaggaac aaatacttct 23460

aaccaggttg ctgttcttta tcaggatgtt aactgcacag aagtccctgt tgctattcat 23520

gcagatcaac ttactcctac ttggcgtgtt tattctacag gttctaatgt ttttcaaaca 23580

cgtgcaggct gtttaatagg ggctgaatat gtcaacaact catatgagtg tgacataccc 23640

attggtgcag gtatatgcgc tagttatcag actcagacta attctcctcg gcgggcacgt 23700

agtgtagcta gtcaatccat cattgcctac actatgtcac ttggtgcaga aaattcagtt 23760

gcttactcta ataactctat tgccataccc acaaatttta ctattagtgt taccacagaa 23820

attctaccag tgtctatgac caagacatca gtagattgta caatgtacat ttgtggtgat 23880

tcaactgaat gcagcaatct tttgttgcaa tatggcagtt tttgtacaca attaaaccgt 23940

gctttaactg gaatagctgt tgaacaagac aaaaacaccc aagaagtttt tgcacaagtc 24000

aaacaaattt acaaaacacc accaattaaa gattttggtg gttttaattt ttcacaaata 24060

ttaccagatc catcaaaacc aagcaagagg tcatttattg aagatctact tttcaacaaa 24120

gtgacacttg cagatgctgg cttcatcaaa caatatggtg attgccttgg tgatattgct 24180

gctagagacc tcatttgtgc acaaaagttt aacggcctta ctgttttgcc acctttgctc 24240

acagatgaaa tgattgctca atacacttct gcactgttag cgggtacaat cacttctggt 24300

tggacctttg gtgcaggtgc tgcattacaa ataccatttg ctatgcaaat ggcttatagg 24360

tttaatggta ttggagttac acagaatgtt ctctatgaga accaaaaatt gattgccaac 24420

caatttaata gtgctattgg caaaattcaa gactcacttt cttccacagc aagtgcactt 24480

ggaaaacttc aagatgtggt caaccaaaat gcacaagctt taaacacgct tgttaaacaa 24540

cttagctcca attttggtgc aatttcaagt gttttaaatg atatcctttc acgtcttgac 24600

aaagttgagg ctgaagtgca aattgatagg ttgatcacag gcagacttca aagtttgcag 24660

acatatgtga ctcaacaatt aattagagct gcagaaatca gagcttctgc taatcttgct 24720

gctactaaaa tgtcagagtg tgtacttgga caatcaaaaa gagttgattt ttgtggaaag 24780

ggctatcatc ttatgtcctt ccctcagtca gcacctcatg gtgtagtctt cttgcatgtg 24840

acttatgtcc ctgcacaaga aaagaacttc acaactgctc ctgccatttg tcatgatgga 24900

aaagcacact ttcctcgtga aggtgtcttt gtttcaaatg gcacacactg gtttgtaaca 24960

caaaggaatt tttatgaacc acaaatcatt actacagaca acacatttgt gtctggtaac 25020

tgtgatgttg taataggaat tgtcaacaac acagtttatg atcctttgca acctgaatta 25080

gactcattca aggaggagtt agataaatat tttaagaatc atacatcacc agatgttgat 25140

ttaggtgaca tctctggcat taatgcttca gttgtaaaca ttcaaaaaga aattgaccgc 25200

ctcaatgagg ttgccaagaa tttaaatgaa tctctcatcg atctccaaga acttggaaag 25260

tatgagcagt atataaaatg gccatggtac atttggctag gttttatagc tggcttgatt 25320

gccatagtaa tggtgacaat tatgctttgc tgtatgacca gttgctgtag ttgtctcaag 25380

ggctgttgtt cttgtggatc ctgctgcaaa tttgatgaag acgactctga gccagtgctc 25440

aaaggagtca aattacatta cacataaacg aacttatgga tttgtttatg agaatcttca 25500

caattggaac tgtaactttg aagcaaggtg aaatcaagga tgctactcct tcagattttg 25560

ttcgcgctac tgcaacgata ccgatacaag cctcactccc tttcggatgg cttattgttg 25620

gcgttgcact tcttgctgtt tttcagagcg cttccaaaat cataaccctc aaaaagagat 25680

ggcaactagc actctccaag ggtgttcact ttgtttgcaa cttgctgttg ttgtttgtaa 25740

cagtttactc acaccttttg ctcgttgctg ctggccttga agcccctttt ctctatcttt 25800

atgctttagt ctacttcttg cagagtataa actttgtaag aataataatg aggctttggc 25860

tttgctggaa atgccgttcc aaaaacccat tactttatga tgccaactat tttctttgct 25920

ggcatactaa ttgttacgac tattgtatac cttacaatag tgtaacttct tcaattgtca 25980

ttacttcagg tgatggcaca acaagtccta tttctgaaca tgactaccag attggtggtt 26040

atactgaaaa atgggaatct ggagtaaaag actgtgttgt attacacagt tacttcactt 26100

cagactatta ccagctgtac tcaactcaat tgagtacaga cactggtgtt gaacatgtta 26160

ccttcttcat ctacaataaa attgttgatg agcctgaaga acatgtccaa attcacacaa 26220

tcgacggttc atccggagtt gttaatccag taatggaacc aatttatgat gaaccgacga 26280

cgactactag cgtgcctttg taagcacaag ctgatgagta cgaacttatg tactcattcg 26340

tttcggaaga gacaggtacg ttaatagtta atagcgtact tctttttctt gctttcgtgg 26400

tattcttgct agttacacta gccatcctta ctgcgcttcg attgtgtgcg tactgctgca 26460

atattgttaa cgtgagtctt gtaaaacctt ctttttacgt ttactctcgt gttaaaaatc 26520

tgaattcttc tagagttcct gatcttctgg tctaaacgaa ctaaatatta tattagtttt 26580

tctgtttgga actttaattt tagccatggc agattccaac ggtactatta ccgttgaaga 26640

gcttaaaaag ctccttgaac aatggaacct agtaataggt ttcctattcc ttacatggat 26700

ttgtcttcta caatttgcct atgccaacag gaataggttt ttgtatataa ttaagttaat 26760

tttcctctgg ctgttatggc cagtaacttt agcttgtttt gtgcttgctg ctgtttacag 26820

aataaattgg atcaccggtg gaattgctat cgcaatggct tgtcttgtag gcttgatgtg 26880

gctcagctac ttcattgctt ctttcagact gtttgcgcgt acgcgttcca tgtggtcatt 26940

caatccagaa actaacattc ttctcaacgt gccactccat ggcactattc tgaccagacc 27000

gcttctagaa agtgaactcg taatcggagc tgtgatcctt cgtggacatc ttcgtattgc 27060

tggacaccat ctaggacgct gtgacatcaa ggacctgcct aaagaaatca ctgttgctac 27120

atcacgaacg ctttcttatt acaaattggg agcttcgcag cgtgtagcag gtgactcagg 27180

ttttgctgca tacagtcgct acaggattgg caactataaa ttaaacacag accattccag 27240

tagcagtgac aatattgctt tgcttgtaca gtaagtgaca acagatgttt catctcgttg 27300

actttcaggt tactatagca gagatattac taattattat gaggactttt aaagtttcca 27360

tttggaatct tgattacatc ataaacctca taattaaaaa tttatctatt gaattgtgcg 27420

tggatgaggc tggttctaaa tcacccattc agtacatcga tatcggtaat tatacagttt 27480

cctgtttacc ttttacaatt aattgccagg aacctaaatt gggtagtctt gtagtgcgtt 27540

gttcgttcta tgaagacttt ttagagtatc atgacgttcg tgttgtttta gatttcatct 27600

aaacgaacaa actaaaatgt ctgataatgg accccaaaat cagcgaaatg caccccgcat 27660

tacgtttggt ggaccctcag attcaactgg cagtaaccag aatggagaac gcagtggggc 27720

gcgatcaaaa caacgtcggc cccaaggttt acccaataat actgcgtctt ggttcaccgc 27780

tctcactcaa catggcaagg aagaccttaa attccctcga ggacaaggcg ttccaattaa 27840

caccaatagc agtccagatg accaaattgg ctactaccga agagctacca gacgaattcg 27900

tggtggtgac ggtaaaatga aagatctcag tccaagatgg tatttctact acctaggaac 27960

tgggccagaa gctggacttc cctatggtgc taacaaagac ggcatcatat gggttgcaac 28020

tgagggagcc ttgaatacac caaaagatca cattggcacc cgcaatcctg ctaacaatgc 28080

tgcaatcgtg ctacaacttc ctcaaggaac aacattgcca aaaggcttct acgcagaagg 28140

gagcagaggc ggcagtcaag cctcttctcg ttcctcatca cgtagtcgca acagttcaag 28200

aaattcaact ccaggcagca gtaggggaac ttctcctgct agaatggctg gcaatggcgg 28260

tgatgctgct cttgctttgc tgctgcttga cagattgaac cagctcgaga gcaaaatgtc 28320

tggtaaaggc caacaacaac aaggccaaac tgtcactaag aaatctgctg ctgaggcttc 28380

taagaagcct cggcaaaaac gtactgccac taaagcatac aatgtaacac aagctttcgg 28440

cagacgtggt ccagaacaaa cccaaggaaa ttttggggac caggaactaa tcagacaagg 28500

aactgattac aaacattggc cgcaaattgc acaatttgcc cccagcgctt cagcgttctt 28560

cggaatgtcg cgcattggca tggaagtcac accttcggga acgtggttga cctacacagg 28620

tgccatcaaa ttggatgaca aagatccaaa tttcaaagat caagtcattt tgctgaataa 28680

gcatattgac gcatacaaaa cattcccacc aacagagcct aaaaaggaca aaaagaagaa 28740

ggctgatgaa actcaagcct taccgcagag acagaagaaa cagcaaactg tgactcttct 28800

tcctgctgca gatttggatg atttctccaa acaattgcaa caatccatga gcagtgctga 28860

ctcaactcag gcctaaactc atgcagacca cacaaggcag atgggctata taaacatttt 28920

cgcttttccg tttacgatat atagtctact cttgtgcaga atgaattctc gtaactacat 28980

agcacaagta gatgtagtta actttaatct cacatagcaa tctttaatca gtgtgtaaca 29040

ttagggagga cttgaaagag ccaccacatt ttcaccgagg ccacgcggag tacgatcgag 29100

tgtacagtga acaatgctag ggagagctgc ctatatggaa gagccctaat gtgtaaaatt 29160

aattttagta gtgctatccc catgtgattt taatagcttc ttaggagaat gacaaaaa 29218

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ccagatgatt ttacaggctg cgtta 25

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgtcaagaat ctcaagtgtc tgtggatc 28

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgagggtcc aaccaacaga gagc 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagttcaca cacttgttct tcac 24

Claims (8)

1. An isolated novel coronavirus having the accession number: CCTCC NO: V202107.

2. Use of the virus of claim 1 for the preparation of milk or serum containing positive antibodies to novel coronaviruses.

3. The use of claim 2, wherein the novel coronavirus is prepared into an immunizing agent and then used for immunizing a cow, and the novel coronavirus is obtained by centrifuging a novel coronavirus liquid which is qualified in inactivation verification to remove cell debris, and then concentrating the solution to obtain an antigen with high concentration;

preparation of an aqueous phase: adding 2% -4% of tween 80 into the concentrated antigen;

preparing an oil phase: adding 5-6% span 85 and 1-2% aluminum stearate into macroro 152 white oil, and sterilizing

And mixing the water phase and the oil phase according to the proportion of 3: 2-4: 3 to obtain the new coronavirus immune preparation.

4. The use of milk or serum prepared as described in claim 2 for evaluating a novel coronavirus animal model, which is constructed by infecting a mouse with SARS-CoV-2/WBP-1, wherein the SARS-CoV-2/WBP-1 has a collection number of CCTCC NO: v202031.

5. Use of milk prepared by the use of claim 1 in the preparation of a drinkable medicament for neutralizing novel coronaviruses.

6. Use of milk or serum prepared by the use as claimed in claim 1 for the preparation of a medicament for the treatment or prevention of a novel coronavirus infection.

7. Use of milk or serum prepared by the use as claimed in claim 1 for the preparation of novel antibodies to coronavirus Igs.

8. The use according to claim 7 of said Igs antibodies for the preparation of a medicament for the treatment or prevention of a novel coronavirus infection.

Images