CN111411118A - Anti-pathogen hyperimmune yolk antibody and AAV vector-based vaccine preparation method and preparation - Google Patents
Anti-pathogen hyperimmune yolk antibody and AAV vector-based vaccine preparation method and preparation Download PDFInfo
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- CN111411118A CN111411118A CN202010244468.0A CN202010244468A CN111411118A CN 111411118 A CN111411118 A CN 111411118A CN 202010244468 A CN202010244468 A CN 202010244468A CN 111411118 A CN111411118 A CN 111411118A
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Abstract
The invention discloses an anti-pathogen hyperimmune yolk antibody, a method for preparing the same based on AAV vector vaccine and a preparation thereof, wherein the method for preparing the hyperimmune yolk antibody comprises the following steps: selecting a first gene sequence containing an antigen protein fragment from a genome sequence of a pathogen, and synthesizing a pathogen protective antigen gene through codon optimization; packaging recombinant AAV vector vaccine carrying antigen gene; immunizing healthy laying hens, and preparing egg yolk antibodies by using eggs of the healthy laying hens. The invention uses the recombinant adeno-associated virus with high safety, quick and simple packaging, low cost and stable expression as the carrier vaccine of protective antigen protein of the expressed virus or bacteria, the egg can be collected by immunizing healthy laying hens once, and the active component in the egg yolk, namely the egg yolk antibody of the virus or bacteria is extracted, and the antibody is a natural immunoglobulin, and can kill the virus or bacteria and prevent the infection of the virus or bacteria.
Description
Technical Field
The invention relates to the technical field of virus vaccines, in particular to an anti-pathogen hyperimmune yolk antibody, and a preparation method and a preparation based on AAV vector vaccines.
Background
Viruses and bacteria have been present in humans on earth for thousands of years, and humans have been immune to many viruses and bacteria. But still has no resistance to some viruses, and the viruses also have mutation and evolution over time, such as influenza viruses which are less harmful to the human body, plagues which are fatal to the human body, and the like. Thus, in order to combat viral attacks, a variety of viral vaccines have emerged. Viral vaccines are typically injections that produce viral antibodies in vivo after one or more injections, thereby protecting against infection by such viruses.
On 11.2.2020, the international committee on viral taxonomy formally named SARS-CoV-2 for the english name of the new coronavirus, and on the same day also formally named 2019 coronavirus pneumonia (COVID-19) by the world health organization.
SARS-CoV-2 belongs to coronavirus belonging to coronavirus family Coronaviridae, is a single-stranded positive-strand RNA virus, and has an envelope on which petal-like or ciliate protrusions are radially arranged, and the shape of the petal-like or ciliate protrusions is similar to that of coronaga, and belongs to β type coronavirus.
The yolk antibody (IgY) is a specific polyclonal antibody existing in immunized poultry eggs, has the molecular weight of 180kDa and is functionally equivalent to mammalian IgG. However, compared to IgG, IgY has many unique advantages:
1) the method is easy to express and obtain in large quantities, and a large amount of IgY can be obtained by collecting eggs after the antigen immunizes the female poultry, so that the method can quickly cope with explosive diseases and play a role in prevention and adjuvant therapy;
2) IgY is relatively stable in nature and does not deactivate at pasteurization temperatures. Acid resistance, no obvious degradation at pH 4.0-11.0, and stable activity at pH less than 3. Experiments show that the IgY lacking the hinge region has the capability of completely avoiding the digestion by trypsin and chymotrypsin in intestinal tracts in vivo, so the IgY can be used for preventing and treating intestinal tract diseases without adding a protective agent;
3) has good safety, and no cross serological reaction between the bird IgY and the mammal immunoglobulin. IgY does not bind with human rheumatic factor or Fc receptor, and does not activate complement system, so it will not cause anaphylaxis;
4) can avoid the generation of false negative or false positive results in the immunoassay process.
Currently, IgY plays an important role in the prevention and control of some human diseases, for example, IgY oral preparations have been successfully applied to the treatment and prevention of infection of gastrointestinal viruses and bacteria such as helicobacter pylori, rotavirus, coronavirus, yersinia pestis, escherichia coli, staphylococcus and the like [5-9], IgY sprays are applied to oral and throat mucous membranes to prevent and treat respiratory tract disease infection, and IgY is also applied to the diagnosis of human tumor diseases. The yolk antibody IgY aiming at SARS-CoV can effectively neutralize SARS-CoV. The 2019 new coronavirus (SARS-CoV-2) has high homology with SARS-CoV virus, and it is suggested that the prepared yolk antibody against SARS-CoV-2 can be used for preventing infection of new coronavirus, clearing virus in gastrointestinal tract, blocking the transmission of feces-mouth, and preventing secondary infection caused by excrement.
The traditional yolk antibody is mainly prepared by taking inactivated pathogen or pathogen protective antigen protein as vaccine, immunizing a mother poultry for many times, and harvesting poultry eggs.
The new coronavirus infection is extremely high, the vaccine development still needs time due to the long period, the public faces the threat of new coronavirus infection for a long time, and the egg yolk antibody aiming at the new coronavirus can kill the new coronavirus to a certain extent, so that a certain infection prevention effect is achieved. The traditional yolk antibody is mainly prepared by taking inactivated pathogen or pathogen protective antigen protein as vaccine, immunizing a mother poultry for many times, and harvesting poultry eggs. The preparation method has the following defects:
(1) on one hand, the workload is increased due to multiple immunizations, so that the labor, material and time costs are increased; on the other hand, the egg laying of the hens is reduced, the antibody level is reduced, even the hens die, and finally the egg yolk antibody titer is reduced;
(2) inactivating pathogens presents a safety risk. By using the inactivated pathogen, the risk of incomplete inactivation and toxin dispersion finally exists, and the risk of pathogen gene recombination also exists in the inactivated pathogen genome;
(3) protein immunization does not continuously stimulate the immune system. By using the pathogenic protective antigen protein, although there is no safe risk of toxin dispersion, the protein is easily decomposed by the organism, and the immune system can not be stimulated to generate antibodies continuously, so that the titer of the yolk antibody is reduced finally.
In view of the above, there is a need for the safe, fast and efficient preparation of a highly immune yolk antibody against a novel coronavirus, which can continuously express an antigen by one-time immunization and can be obtained safely, fast and on a large scale, and a method thereof.
Disclosure of Invention
The invention aims to solve the technical problems that the prior high-immunity yolk antibody for resisting the new coronavirus can not be obtained by one-time immunization, the safety risk exists in pathogen inactivation, and the protein immunization can not continuously stimulate an immune system.
In order to solve the technical problems, the technical scheme adopted by the invention is to provide a method for preparing an anti-pathogen hyperimmune yolk antibody based on an AAV vector vaccine, which comprises the following steps:
selecting a first gene sequence containing an antigen protein fragment from a genome sequence of the pathogen, and synthesizing a corresponding pathogen protective antigen gene through chicken codon optimization;
obtaining a vector fragment by double restriction of a core plasmid pAAV-CMV-eGFP of the AAV vector vaccine; amplifying to obtain a second gene sequence with two ends containing double enzyme cutting sites by using an antigen protein amplification primer of the antigen gene, carrying out double enzyme cutting on the second gene sequence again, recovering, and transfecting with the vector fragments respectively to obtain a recombinant AAV vector vaccine carrying the antigen protein;
selecting healthy laying hens, inoculating the recombinant AAV vector vaccine to breast muscle of the hens at multiple points, and collecting eggs of the hens after immunization;
and preparing an anti-pathogen hyperimmune yolk antibody aiming at the pathogen by using the yolk liquid of the chicken eggs.
In another preferred embodiment, the pathogen is a new coronavirus, and the step of obtaining a protective antigen gene of the pathogen comprises:
selecting a spike glycoprotein S1 fragment gene sequence containing an RBD structural domain from a genome sequence of the new coronavirus, and synthesizing a corresponding pathogen protective antigen gene by chicken codon optimization.
In another preferred embodiment, the steps of obtaining a recombinant AAV vector vaccine carrying antigenic proteins are specifically:
carrying out double enzyme digestion on the core plasmid pAAV-CMV-eGFP by using EcoRI and BamHI, and recovering and obtaining a vector fragment AAV-CMV;
respectively designing amplification primers of antigen genes of spike glycoprotein S1 and RBD, using an S1 gene fragment of a new coronavirus as a template, amplifying to obtain an S1 antigen gene fragment and an RBD antigen gene fragment of which two ends contain EcoRI and BamHI enzyme cutting sites, performing double enzyme cutting recovery by using EcoRI and BamHI, connecting with a vector fragment AAV-CMV, and transforming DH5 α competence to obtain a first core plasmid pAAV-CMV-S1 and a second core plasmid pAAV-CMV-RBD;
extracting the first core plasmid pAAV-CMV-S1 and the second core plasmid pAAV-CMV-RBD without endotoxin, and mixing the extracts with two auxiliary plasmids according to a molar ratio of 1: 1: 1, co-transfecting HEK293T cells with the cationic transfection reagent PEI;
collecting the transfected HEK293T cells, repeatedly freezing and thawing for multiple times, collecting supernate, and adding nuclease for incubation;
and after incubation, collecting supernatant again, and removing impurity proteins and impurities to obtain the recombinant AAV vector vaccine rAAV-S1 and rAAV-RBD.
In another preferred embodiment, the step of immunocapturing the chicken eggs is specifically as follows:
selecting healthy hens, inoculating the recombinant AAV vector vaccine rAAV-S1 and the rAAV-RBD to thoracic muscles of the hens at multiple points according to the dose of 1E +11 vg/hen, and collecting hen eggs after immunization;
disinfecting the eggshell of the egg, selecting the eggshell which is seriously polluted in advance to be disinfected separately, washing the eggshell by clear water, soaking the eggshell and the cleaned eggshell into 0.1 percent benzalkonium bromide water solution with the water temperature of 42 ℃ to disinfect for 15min, then soaking the eggshell and the cleaned eggshell into 90-95 ℃ water bath to disinfect for 5 seconds, taking out the eggshell quickly, airing the eggshell, and storing the eggshell at the temperature of 4 ℃.
In another preferred embodiment, the yolk antibody is obtained by ammonium sulfate precipitation, specifically comprising the steps of:
diluting the yolk with deionized water by 7 times, adjusting the pH value to 5.2 with 3m L HCl solution with the concentration of 0.1 mol/L, standing for 6h at 4 ℃, centrifuging for 30min at 12000r/min, and filtering to obtain a supernatant;
taking 3 parts of 2m L supernatant respectively, adding saturated ammonium sulfate solution until the final concentration is 40%, 60% and 80%, standing overnight at 4 ℃, centrifuging at 5000r/min for 25min, discarding the supernatant, and re-dissolving the precipitate with 2m L pure water respectively.
In another preferred embodiment, the yolk antibody is obtained by a polyethylene glycol precipitation method, which comprises the following steps:
adding 20m L Tris-HC L buffer solution into the yolk, wherein the concentration of the Tris-HC L buffer solution is 0.1 mol/L, the pH value is 7.6, adding PEG after uniformly mixing until the concentration W/V is 3.5%, fully mixing, and centrifuging at 8000r/min for 25 min;
adding PEG into the supernatant until the final concentration W/V is 12%, mixing thoroughly, centrifuging at 12000r/min for 10min, and discarding the supernatant;
the precipitate was redissolved in 10m L of 1 × PBS, PEG was added to a final concentration of 12% W/V, mixed well, centrifuged at 12000r/min for 10min, the supernatant was discarded, and the precipitate was redissolved in 2m L of PBS.
In another preferred embodiment, the yolk antibody is obtained by cold ethanol precipitation, which comprises the following steps:
diluting the yolk with deionized water by 7 times, adjusting the pH value to 5.2 with 3m L HCl solution with the concentration of 0.1 mol/L, standing for 6h at 4 ℃, centrifuging for 30min at 12000r/min, and filtering to obtain a supernatant;
adding 95% ethanol to a final concentration of V/V to 60%, stirring at 4 deg.C for 30min, and centrifuging at 4 deg.C at 10000r/min for 30min to obtain precipitate;
dissolving the precipitate in 10m L NaCl solution with concentration of 0.028 mol/L, filtering to remove lipid at room temperature, adding 95% ethanol into the filtrate to make the final concentration V/V to 30%, mixing well, centrifuging at 4 deg.C at 10000r/min for 30min, and dissolving the precipitate in 2m L PBS solution.
In another preferred embodiment, the yolk antibody is obtained by a water dilution method, which comprises the following steps:
diluting the yolk with deionized pure water, adjusting pH to 7.6, stirring at 4 deg.C for 4 hr, centrifuging, discarding precipitate, filtering the supernatant with 200KDa filter membrane, and concentrating with 150KDa protein ultrafiltration tube to 2 ml.
The invention also discloses an anti-pathogen hyperimmune yolk antibody prepared based on the AAV vector vaccine, and the preparation method is adopted.
The invention also discloses an anti-pathogen hyperimmune yolk antibody spray preparation prepared based on the AAV vector vaccine, which is prepared by adopting the anti-pathogen hyperimmune yolk antibody.
Compared with the prior art, the invention has the following advantages:
1. the eggs can be collected by immunizing healthy laying hens for one time, and active ingredients (anti-virus or anti-bacterial egg yolk antibodies) in the egg yolk are extracted, so that the workload is greatly reduced, and the labor, material and time costs are reduced; and moreover, the stress of the female poultry is not easily caused, and the individual health of the female poultry and the effectiveness of the egg yolk antibody are effectively ensured.
2. Recombinant adeno-associated virus (rAAV) with high safety, rapid and simple packaging, low cost and stable expression is used as a vector vaccine for expressing pathogen protective antigen genes of viruses or bacteria, and inactivated pathogens are not adopted, so that the safety is high, and the risk of virus dispersion is avoided; moreover, the host range of the adeno-associated virus is very wide, the tissue infection capacity is strong, the recombinant adeno-associated virus deletes the virus structure of wild AAV, only the Cap protein, Rep protein and reverse complementary sequences ITRs at the two ends are left, the foreign gene can be efficiently expressed, a large amount of soluble recombinant protein with better antigenicity and immunogenicity and similar functions to the natural protein can be obtained, the protein is not easy to be decomposed by organisms, the foreign gene can be expressed for a long time, and the active division capacity of host cells is not depended on, so that the immune system can be continuously stimulated, and effective yolk antibody can be finally obtained.
3. The method for preparing the egg yolk antibody is safe, efficient, rapid, low in cost, high in extraction efficiency, high in yield and easy to industrialize, and is an important means for preventing and controlling major outbreak epidemic situations.
4. The yolk antibody for resisting virus or bacteria adopts an ultraviolet spectrophotometer to determine that the protein concentration is 18-30 mg/ml; the antigen can be combined with pathogen protective antigen genes of viruses or bacteria through indirect immunofluorescence detection, namely, the aerosol can be prepared into a spray preparation for preventing infection caused by the viruses or the bacteria.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a map of the core plasmid pAAV-CMV-eGFP of the AAV vector vaccine of the present invention;
FIG. 3 is a diagram showing a gel electrophoresis of the double-enzymatic cleavage of the core plasmid pAAV-CMV-eGFP of the present invention with EcoRI and BamHI;
FIG. 4 is a gel electrophoresis diagram of the amplification of the S1 gene according to the present invention;
FIG. 5 is a gel electrophoresis diagram of the amplification of RBD gene according to the present invention;
FIG. 6 is a map of the first core plasmid pAAV-CMV-S1 in the present invention;
FIG. 7 is a map of a second core plasmid pAAV-CMV-RBD in the present invention;
FIG. 8 is a SDS-PAGE gel of the yolk antibody of S1 against the novel coronavirus SARS-CoV2 according to the present invention;
FIG. 9 is a SDS-PAGE gel of an RBD protein yolk antibody against the novel coronavirus SARS-CoV2 according to the present invention;
FIG. 10 is an indirect immunofluorescence of the S1 protein yolk antibody against the novel coronavirus SARS-CoV2 in the present invention;
FIG. 11 is an indirect immunofluorescence of RBD protein yolk antibody against the novel coronavirus SARS-CoV2 in the present invention;
FIG. 12 is the green fluorescence expression diagram of DF1 cells infected by rAAV-CMV-GEP in the present invention;
FIG. 13 is a green fluorescence expression pattern of rAAV-CMV-GEP-infected hen leg muscle in the present invention.
Detailed Description
It has been proved by present research that SARS-CoV-2, like SARS-CoV, mediates the invasion of virus into host cell by the combination of Spike protein on virus surface and angiotensin converting enzyme 2 (ACE2) as host cell surface receptor, but it has also been proved that IgG aiming at SARS full-length Spike protein can mediate SARS virus to infect macrophage and thus to aggravate infection.
Therefore, the invention provides an anti-pathogen hyperimmune yolk antibody prepared based on AAV vector vaccine, a method and a preparation, which are used for preparing the yolk antibody aiming at the Spike protein of SARS-CoV-2 virus for preventing the infection of new coronavirus. It is more advantageous to use partial fragment of Spike protein of SARS-CoV-2 virus, such as S1 protein or receptor-binding domain (RBD) gene, as antigen to prepare the yolk antibody. The invention is described in detail below with reference to the drawings and the detailed description.
The invention provides a method for preparing an anti-pathogen hyperimmune yolk antibody based on an AAV vector vaccine, which comprises the following steps:
according to the genome sequence of the pathogen (virus or bacteria), selecting a first gene sequence containing an antigen protein fragment, and synthesizing a corresponding pathogen protective antigen gene through chicken codon optimization;
carrying out double digestion on core plasmid pAAV-CMV-eGFP of AAV vector vaccine (adeno-associated virus vector vaccine) to obtain a vector fragment; aiming at the pathogen protective antigen gene synthesized by optimizing chicken codons, designing an antigen protein amplification primer, amplifying a gene segment of a pathogen by using the amplification primer to obtain a second gene sequence with double enzyme cutting sites at two ends, carrying out double enzyme cutting on the pAAV-CMV-antigen gene of the second gene sequence, respectively transfecting with the vector segment after recovery to obtain a recombinant AAV vector vaccine rAAV-antigen gene carrying the antigen protein, determining the titer of the rAAV-antigen gene by adopting fluorescent quantitative PCR, and subpackaging and storing;
selecting healthy laying hens, inoculating the recombinant AAV vector vaccine (rAAV-antigen gene) obtained in the previous step to breast muscles of the hens at multiple points, collecting eggs after 20 days of immunization, sterilizing eggshells, and storing;
opening the egg, draining off the egg white, absorbing the residual egg white by using filter paper, uniformly mixing egg yolk liquid, putting 10m of L egg yolk liquid into a centrifugal tube, and preparing the anti-pathogen hyperimmune egg yolk antibody aiming at the pathogen by using the egg yolk liquid of the egg.
According to the invention, the recombinant adeno-associated virus with high safety, rapid and simple packaging, low cost and stable expression is used as a vector vaccine for expressing protective antigen genes of viruses or bacteria, and the eggs can be collected by immunizing a healthy laying hen for one time, so that the working efficiency is effectively improved.
The adeno-associated virus vector is used as a vector applied to human clinical gene therapy, has the advantages of high safety, relatively simple virus packaging operation, low cost, stable expression and the like, and is commonly used as a gene engineering live vaccine vector. The host range is very wide, the tissue infection capacity is strong, and the exogenous gene can be expressed for a long time without depending on the active division capacity of host cells. The recombinant adeno-associated virus (rAAV) deletes the virus structure of wild AAV, only leaves the Cap protein, Rep protein and reverse complementary sequences ITRs at both ends, can not only express exogenous gene with high efficiency, but also obtain a large amount of soluble recombinant protein with better antigenicity and immunogenicity and similar function to natural protein.
Detailed description of the preferred embodiments
The virus or bacterium is a new coronavirus, as shown in figure 1.
And S10, synthesizing a pathogen protective antigen gene.
The genome sequence (ID: MN988668.1) of the new coronavirus SARS-CoV2 is obtained from GeneBank, the gene sequence of the spike glycoprotein S1 fragment (containing RBD structural domain) is selected, and the gene sequence is sent to a gene synthesis company after the codon optimization of the chicken, so as to synthesize the corresponding pathogen protective antigen gene.
S20, packaging the recombinant AAV vector vaccine carrying the S1 or RBD gene, which specifically comprises the following steps:
step S21, constructing a first core plasmid pAAV-CMV-S1 and a second core plasmid pAAV-CMV-RBD.
The core plasmid pAAV-CMV-eGFP (the map of which is shown in figure 2) is subjected to double enzyme digestion by EcoRI and BamHI (the double enzyme digestion gel electrophoresis map of which is shown in figure 3), the carrier fragment AAV-CMV is recovered, spike glycoprotein S1 and RBD amplification primers are respectively designed, a new coronavirus S1 gene is used as a template, an S1 gene fragment and an RBD gene fragment which contain EcoRI and BamHI enzyme digestion site sequences at two ends are obtained by amplification, the S1 gene and RBD gene amplification gel electrophoresis maps are respectively shown in figures 4 and 5, after double enzyme digestion recovery is carried out by EcoRI and BamHI, the obtained product is connected with the carrier fragment AAV-CMV and then transformed into a DH5 α sensitive state, and sequencing and identification are carried out, so that a first core plasmid pAAV-CMV-S1 (the map of which is shown in figure 6) and a second core plasmid pAAV-CMV-RBD (the map of which is shown in figure 7) are obtained.
Specifically, the gene sequence of the first core plasmid pAAV-CMV-S1 is: ctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcg acctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatca ctaggggttcctgcggccgcacgcgtggagctagttattaatagtaatcaattacggggtcatt agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctga ccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatca agtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcat tatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg ctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacg gggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacggg actttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtg ggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgc tgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccgg gaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtct ataggcccacaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatact ttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattct aaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttct gcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctacc attctgcttttattttatggttgggataaggctggattattctgagtccaagctaggccctttt gctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgtg ctggcccatcactttggcaaagaattgggattcgaacatcgattgaattcgccaccatgtttgt gtttctggtgctgctgcccctggtgagcagccagtgcgtgaatctgacaacaaggacccagctg ccccccgcctacaccaacagtttcacaagaggcgtgtactaccctgataaagtgtttagaagca gcgtgctgcactccacacaggatctgtttctgccctttttctccaatgtgacctggttccatgc tattcacgtgtctggaacaaacggcacaaaacggttcgataaccccgtgctgcccttcaacgac ggagtgtacttcgcatccactgagaaatcaaatatcattcgggggtggatctttggcacaacac tggatagcaaaacccagagcctgctgatcgtgaacaatgcaacaaacgtggtcattaaggtgtg cgagtttcagttttgcaacgatccattcctgggcgtgtactaccacaagaataataagagctgg atggaaagcgagtttagagtgtacagctctgccaacaactgcacctttgagtacgtgtcccagc ctttcctgatggatctggaaggcaaacagggcaactttaaaaacctgcgcgagttcgtctttaa gaacattgatggctacttcaagatctattccaaacacacaccaatcaatctggtgagagatctg ccacagggattttcagcactggagcccctggtggatctgcctattggaatcaatatcacaaggt tccagacactgctggctctgcacagaagctacctgacccctggagattcttccagcgggtggac cgccggagccgccgcctactacgtgggatacctgcagccaagaaccttcctgctgaaatataac gaaaacggaacaattactgatgccgtggactgcgccctcgacccactgagcgaaactaaatgca ccctgaagagcttcaccgtggagaagggcatctatcagacatctaacttcagggtgcagcccac cgaatctattgtgaggtttcccaacatcactaacctgtgcccattcggcgaggtgtttaacgct actaggtttgctagcgtgtacgcatggaaccgcaagagaattagcaattgtgtggccgattata gcgtcctgtataacagcgcctctttcagcacattcaagtgctacggcgtgtcccctaccaagct gaatgatctgtgcttcacaaacgtgtatgctgatagctttgtgattagaggcgacgaggtgcgg cagatcgctcctggccagaccggcaagatcgctgactacaactacaagctgcccgatgacttca caggctgcgtgatcgcttggaactccaacaacctggattccaaggtgggcggcaactacaatta tttgtacagactgtttaggaaaagcaacctgaagccctttgaaagagacattagcacagagatc taccaggccgggtctactccttgcaacggcgtcgaaggctttaattgttacttcccactgcaga gctacggattccagcccactaacggagtggggtaccagccataccgcgtggtggtgctgtcctt tgaattgctgcatgcccccgctacagtgtgcgggcccaagaagtccactaacctggtgaagaataagtgcgtgaacttcaattttaacggcctgaccggaacaggcgtgctgactgaatctaacaaga agttcctgccatttcagcagttcgggagggacatcgctgacaccactgatgccgtgagagaccc ccagaccctggagattctggatatcactccatgcagctttggcggcgtgagcgtgatcacaccc ggcacaaacacctctaatcaggtggcagtgctgtaccaggacgtgaactgcacagaggtgcccg tggccattcacgctgatcagctgacccccacctggagagtgtacagcaccggaagcaacgtgtt ccagacaagagccggctgcctgatcggagccgagcatgtgaacaacagctacgagtgcgatatt cccatcggcgctggcatctgcgctagctaccagtacccctacgacgtgcccgactacgcctgag gatcctctagagtcgacctgcagaagcttgcctcgagcagcgctgctcgagagatctacgggtg gcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccacc agccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatatta tggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcgg ggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcc tgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgacca ggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctc caactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtga accactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgca ggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccggg cgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca gctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg catacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtgg ttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccc ttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttaggg ttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgta gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatag tggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataa gggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcga attttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgc cgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctg ctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggtttt caccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaa tgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacc cctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgat aaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttat tcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaa gatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaaga tccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatg tggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattct cagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaa gagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaac gatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcctt gatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctg tagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccg gctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcag cactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaac tatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactg tcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgta atctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagc taccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttct agtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaa gacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccag cttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacg cttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgca cgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctg acttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac gcggcctttttacggttcctggccttttgctggccttttgctcacatgt
The gene sequence of the second core plasmid pAAV-CMV-RBD is as follows: cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggc gacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatc actaggggttcctgcggccgcacgcgtggagctagttattaatagtaatcaattacggggtcat tagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgtcaata gggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggca ttatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcac ggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggt gggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacg ctgttttgacctccatagaagacaccgggaccgatccagcctccgcggattcgaatcccggccg ggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagagtc tataggcccacaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatac tttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttgcaccattc taaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttc tgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctac cattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttt tgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaacgtgctggtctgtgt gctggcccatcactttggcaaagaattgggattcgaacatcgattgaattcgccaccatgtcca tgttccgcgtgcagccaaccgagagcatcgtgcggtttccaaacattacaaatctgtgcccctt cggggaagtgtttaacgcaacaagatttgccagcgtgtacgcttggaacagaaagaggatttcc aattgcgtggctgattacagcgtgctgtacaatagcgcatccttcagcacttttaagtgctacg gcgtgtcccccactaaactgaacgacctgtgcttcaccaacgtgtacgccgatagctttgtgat cagaggagacgaagtgaggcagattgcacctggacagactgggaagatcgccgattacaactac aaactgccagatgacttcaccggatgcgtgatcgcttggaacagcaacaatctggacagcaaag tgggcggaaactacaattacctgtaccgcctgttccggaagtccaacctgaaaccctttgagag agatatctccacagaaatttaccaggcaggaagcactccttgcaacggcgtggaaggattcaat tgctactttcctctgcagagctacgggtttcagccaaccaacggggtgggctaccagccctaca gggtgtacccctacgatgtgcctgactacgcctgaggatcctctagagtcgacctgcagaagct tgcctcgagcagcgctgctcgagagatctacgggtggcatccctgtgacccctccccagtgcct ctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgca tcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaa ggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgca gtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagc ctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggta gagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctaccca ccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctga ttttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccact ccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggta ttttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgc cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgc cagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggcttt ccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcg accccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttt tcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca ctcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggt taaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaat tttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccg ccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacg aaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacg tcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaa gagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcct gtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgag tgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacg ttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgcc gggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccag tcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccat gagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgct tttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaag ccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctg gagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccg tatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgct gagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttt agattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatct catgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactgg cttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttc aagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcg gtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggt atccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctg gtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcg tcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcctttt gctggccttttgctcacatgt
The amplification primer F of S1 is: 5'-CCGGAATTCGCCACCATGTTTGTGTTTCTGGTGCTG-3', R: 5'-CGCGGATCCTCAGGCGTAGTCGGGCACGTC-3'
The RBD amplification primer F is: 5'-CCGGAATTCGCCACCATGTCCATGTTCCGCGTGCAG-3', R: 5'-CGCGGATCCTCAGGCGTAGTCAGGCACATC-3'
And S22, co-transfecting three plasmids to package the rAAV-S1 and the rAAV-RBD.
Extracting a first core plasmid pAAV-CMV-S1, a second core plasmid pAAV-CMV-RBD and two auxiliary plasmids without endotoxin, co-transfecting HEK293T cells with a cationic transfection reagent PEI according to a molar ratio of 1: 1: 1, collecting the cells after 72 hours, repeatedly freezing and thawing in a liquid bath at the temperature of liquid nitrogen/37 ℃ for four times, centrifuging at the temperature of 4 ℃ for 10min by 10000g, collecting supernatant containing recombinant adeno-associated virus, adding nuclease into the supernatant to a final concentration of 50U/m L, mixing, incubating in a water bath at the temperature of 37 ℃ for 1 hour, centrifuging at the temperature of 4 ℃ for 10min by 10000g, collecting supernatant, sequentially removing impurities by using a 0.45 micron and 0.22 micron filter, collecting filtered samples, concentrating by using a 100kD ultrafiltration protein ultrafiltration filter tube, removing impurities such as cell factors, adding PBS (which can be replaced by PBS), quantitatively blocking up a sample by using a Pluronic filter, and quantitatively measuring the titer by using a PCR-82 PCR-ELISA.
Specifically, EcoRI is a restriction enzyme in E.coli, specifically recognizes the sequence of GAATTC, and cleaves it between G and A. BamH I is a restriction enzyme that specifically recognizes the sequence of GGATCC and cleaves it between G and G. PEI transfection reagent is a cationic polymer with a molecular weight of 25000, which is capable of forming a complex with nucleic acid and allowing the complex to enter mammalian cells, and is widely used in common cell lines such as HEK-293, HEK293T, Hep G2, Hela, CHO-K1, COS-1, COS-7, NIH/3T3, Sf9, etc., and it is highly efficient in introducing nucleic acid into cells even in the presence of serum. PBS is phosphate buffered saline (phosphate buffer saline) generally used as a solvent, plays a role of dissolving and protecting a reagent, is the most widely used buffer solution in biochemical research, mainly comprises Na2HPO4, KH2PO4, NaCl and KCl, and has wide pH value range due to secondary dissociation of Na2HPO4 and KH2PO 4; while NaCl and KCl mainly act to increase the salt ion concentration. The fluorescent quantitative PCR is to mark and track the PCR product through a fluorescent dye or a fluorescent-marked specific probe, monitor the reaction process on line in real time, analyze the product by combining with corresponding software, and calculate the initial concentration of a sample template to be detected. HEK293 cells are divided into several types, 293A for adenovirus and 293T for lentivirus, the latter being used in this example.
S30, immunizing healthy laying hens, and collecting eggs of the hens.
S31, selecting healthy white leghorn hens, inoculating rAAV-S1 and rAAV-RBD to breast muscles of the hens at multiple points according to the dose of 1E +11 vg/hen, and collecting hen eggs after 20 days of immunization;
s32, disinfecting eggshells, selecting the eggshells which are seriously polluted, disinfecting the eggshells separately in advance, washing the eggshells with clear water, soaking the eggshells and the cleaned eggshells into 0.1% benzalkonium bromide water solution with the water temperature of 42 ℃ for disinfection for 15min, then soaking the eggshells and the cleaned eggshells into 90-95 ℃ water bath for disinfection for 5 seconds, taking out the eggshells quickly, airing the eggshells, and storing the eggshells at the temperature of 4 ℃.
Specifically, the white leghorn hens lay Italy and distribute a very wide and famous egg variety in the world, and the chicken has small body, early maturity, no nidation, high egg yield and low feed consumption.
Step S40, extracting the yolk antibody
Opening chicken eggs, draining egg white, absorbing residual egg white by using filter paper, uniformly mixing egg yellow liquid, putting 10m of L egg yellow liquid into a centrifugal tube, and obtaining an anti-pathogen hyperimmune egg yolk antibody aiming at spike glycoprotein S1 and RBD structural domain of the neocoronavirus SARS-CoV2 by using an ammonium sulfate precipitation method, a polyethylene glycol precipitation method, a cold ethanol precipitation method or a water dilution method, wherein the egg yolk antibody contains recombinant AAV vector vaccine carrying antigenic protein.
Specifically, the ammonium sulfate precipitation method comprises the following specific steps:
diluting yolk with deionized water 7 times, adjusting pH to 5.2 with 3m L HCl solution with concentration of 0.1 mol/L, standing at 4 deg.C for 6 hr, centrifuging at 12000r/min for 30min, filtering to obtain supernatant;
taking 3 parts of 2m L supernatant respectively, adding saturated ammonium sulfate solution until the final concentration is 40%, 60% and 80%, standing overnight at 4 ℃, centrifuging at 5000r/min for 25min, discarding the supernatant, and re-dissolving the precipitate with 2m L pure water respectively.
Specifically, the polyethylene glycol precipitation method comprises the following specific steps:
adding 20m L concentration 0.1 mol/L Tris-HC L buffer solution with pH value 7.6 into yolk, mixing uniformly, adding PEG to the concentration of 3.5% (W/V) and mixing fully, centrifuging at 000r/min for 25min, taking supernatant, adding PEG to the final concentration of 12% (W/V) and mixing fully, and centrifuging at 12000r/min for 10 min;
discarding supernatant, re-dissolving the precipitate with 10m L PBS 1 ×, adding PEG to final concentration of 12% (W/V), mixing thoroughly, centrifuging at 12000r/min for 10min, discarding supernatant,
the precipitate was redissolved in 10m L of 1 × PBS, PEG was added to a final concentration of 12% W/V, mixed well, centrifuged at 12000r/min for 10min, the supernatant was discarded, and the precipitate was redissolved in 2m L PBS.
Wherein, PEG is the abbreviation of polyethylene glycol in English name of polyethylene glycol, has no toxicity, no irritation, good water solubility and good intermiscibility with a plurality of organic matter components. The PBS solution is also called phosphate buffer solution, and is generally prepared by selecting Na2HPO4 and KH2PO4 because the sodium salt dissolves slowly. According to the solutions with different pH values, phosphate with different qualities is weighed, and the pH value of the solution can also be adjusted by a pH meter.
Specifically, the cold ethanol precipitation method comprises the following specific steps:
diluting yolk with deionized water 7 times, adjusting pH to 5.2 with 3m L HCl solution with concentration of 0.1 mol/L, standing at 4 deg.C for 6 hr, centrifuging at 12000r/min for 30min, filtering to obtain supernatant;
adding 95% cold ethanol to a final concentration of 60% (V/V), stirring at 4 deg.C for 30min, centrifuging at 4 deg.C at 10000r/min for 30min to obtain precipitate;
dissolving the precipitate in 10m L NaCl solution with concentration of 0.028 mol/L, filtering to remove lipid at room temperature, adding 95% cold ethanol into the filtrate to make the final concentration to 30% (V/V), mixing well, centrifuging at 10000r/min for 30min at 4 deg.C, and dissolving the precipitate in 2m L PBS solution.
Specifically, the water dilution method comprises the following specific steps:
diluting yolk with deionized pure water, adjusting pH to 7.6, stirring at 4 deg.C for 4 hr, centrifuging, discarding precipitate, filtering supernatant with 200KDa filter membrane, and concentrating to 2ml with 150KDa protein ultrafiltration tube.
Thus, protective yolk antibodies against the S1 protein and RBD domain of the novel coronavirus SARS-CoV2 were obtained by the above steps.
Second, immunoassay
The yolk antibody against the S1 protein and RBD domain of SARS-CoV2 was subjected to mapping, protein concentration and binding to antigen.
1. Atlas
A yolk antibody (the anti-pathogen hyperimmune yolk antibody prepared by the method) aiming at the S1 protein and the RBD structural domain of SARS-CoV2 is prepared by taking 32 mu L, adding 8 mu L5 × protein loading buffer, bathing in boiling water for 10min, standing on ice for 10min, spotting, carrying out 80V voltage, and carrying out SDS-PAGE electrophoresis for 2h, wherein the electrophoresis images are respectively shown in figures 8 and 9.
Placing into a dyeing bar, pouring improved Coomassie staining solution, placing in a microwave oven with middle fire for 1min, taking out, placing in a horizontal shaking table, shaking for 10min, changing the decolorizing solution, repeating for 2 times, and imaging, respectively as shown in FIG. 8 and FIG. 9.
And (3) detection results: the anti-pathogen hyperimmune yolk antibody aiming at the S1 protein and the RBD structural domain of SARS-CoV2 shows two bands after being stained by improved Coomassie brilliant blue according to SDS-PAGE gel electrophoresis, and two specific bands can be found at 70KDa and 25KDa by WesternBlotting detection.
2. Protein concentration
The protein concentration is measured by ultraviolet spectrophotometer to be 18-30 mg/ml.
3. Binding to antigen
Detection with indirect immunofluorescence:
1) respectively transfecting the pAAV-CMV-S1 and the pAAV-CMV-RBD into HEK293T cells for 48h
2)1 × PBS 3 times, each 3min
3) Fixing with 4% paraformaldehyde for 15min
4)1 × PBS 3 times, each 3min
5) The anti-pathogenic hyperimmune yolk antibodies obtained in Experimental examples 2 and 3 were diluted 200-fold with PBS containing 5% BSA and 0.1% Triton X-100, added to the culture plates, and incubated at 37 ℃ for 2 hours
6)1 × PBS 3 times, each 3min
7) Adding 1 × PBS containing green fluorescence labeled rabbit anti-IgY, and incubating at 37 deg.C for 1h
8)1 × PBS 3 times, each 3min
The fluorescence microscopy images are shown in FIG. 10 and FIG. 11, respectively.
And (3) detection results: it can be combined with protective antigen S1 of new coronavirus SARS-CoV2 by indirect immunofluorescence detection. It can be combined with protective antigen RBD of new coronavirus SARS-CoV2 by indirect immunofluorescence detection.
Third, verify
To verify that the recombinant adeno-associated virus vector can infect chickens and express proteins, verification was performed at the cellular level and at the in vivo level, respectively:
1. cell level validation
The chicken fibroblast cells DF1 are inoculated with recombinant AAV vector rAAV-CMV-eGFP carrying enhanced green fluorescent protein gene with MOI of 100, and the green fluorescent protein expression can be seen by observing the chicken fibroblast cells DF1 after infection for 3 days by using a fluorescence microscope (as shown in figure 12), thereby proving that rAAV can infect chicken cells and express protein.
2. In vivo level verification
The recombinant AVV vector rAAV-CMV-eGFP carrying the enhanced green fluorescent protein gene is used for inoculating leg muscles of healthy white leghorns with 1E +11 vg/dose, after 20 days of infection, the green fluorescent protein expression can be seen by observing a material section by using a fluorescence microscope, and the rAAV can infect the hens and express the protein is proved to be shown in figure 13.
The invention also discloses an anti-pathogen hyperimmune yolk antibody prepared based on the AAV vector vaccine, and the preparation method is adopted.
The invention also discloses an anti-pathogen high-immunity yolk antibody spray preparation prepared based on AAV vector vaccine, which is prepared by mixing 200mg of the high-immunity yolk antibody with 5g of sorbitol, 0.05g of sucralose, 2g of hydrogenated castor oil and 494.78g of deionized water after drying by using a spray dryer by using 0.06% glucose solution as a protective agent. The spraying form is adopted, so that the use is more facilitated, and the operation difficulty is low.
Compared with the prior art, the invention has the following advantages:
1. the eggs can be collected by immunizing healthy laying hens for one time, active ingredients (anti-virus or bacteria hyperimmune egg yolk antibody) in the egg yolk are extracted, the workload is greatly reduced, and the labor, material and time costs are reduced; and moreover, the stress of the female poultry is not easily caused, and the individual health of the female poultry and the effectiveness of the hyperimmune egg yolk antibody are effectively ensured.
2. Recombinant adeno-associated virus (rAAV) with high safety, rapid and simple packaging, low cost and stable expression is used as a vector vaccine for expressing pathogen protective antigen genes of viruses or bacteria, and inactivated pathogens are not used, so that the safety is high, and the risk of virus dispersion is avoided; moreover, the host range of the adeno-associated virus is very wide, the tissue infection capacity is strong, the recombinant adeno-associated virus deletes the virus structure of wild AAV, only the Cap protein, Rep protein and reverse complementary sequences ITRs at the two ends are left, the foreign gene can be efficiently expressed, a large amount of soluble recombinant protein with better antigenicity and immunogenicity and similar functions to the natural protein can be obtained, the protein is not easy to be decomposed by organisms, the foreign gene can be expressed for a long time, and the active division capacity of host cells is not depended on, so that the immune system can be continuously stimulated, and the effective high-immunity yolk antibody can be finally obtained.
3. The method for preparing the high-immunity yolk antibody is safe, efficient, rapid, low in cost, high in extraction efficiency, high in yield and easy to industrialize, and is an important means for preventing and controlling major outbreak epidemic situations.
4. The invention discloses a virus or bacteria resistant hyperimmune yolk antibody, which adopts an ultraviolet spectrophotometer to determine that the protein concentration is 18-30 mg/ml; the antigen can be combined with a virus or bacteria pathogen protective antigen gene through indirect immunofluorescence detection, namely, the antigen is prepared into a spray preparation for preventing infection caused by the virus or bacteria.
The present invention is not limited to the above-mentioned preferred embodiments, and any structural changes made under the teachings of the present invention shall be understood as falling within the protection scope of the present invention.
Claims (10)
1. The method for preparing the anti-pathogen hyperimmune yolk antibody based on the AAV vector vaccine is characterized by comprising the following steps of:
selecting a first gene sequence containing an antigen protein fragment from a genome sequence of the pathogen, and synthesizing a corresponding pathogen protective antigen gene through chicken codon optimization;
obtaining a vector fragment by double restriction of a core plasmid pAAV-CMV-eGFP of the AAV vector vaccine; amplifying to obtain a second gene sequence with two ends containing double enzyme cutting sites by using an antigen protein amplification primer of the antigen gene, carrying out double enzyme cutting on the second gene sequence again, recovering, and transfecting with the vector fragments respectively to obtain a recombinant AAV vector vaccine carrying the antigen protein;
selecting healthy laying hens, inoculating the recombinant AAV vector vaccine to thoracic muscles of the hens at multiple points, and collecting eggs after immunization;
and preparing an anti-pathogen hyperimmune yolk antibody aiming at the pathogen by using the yolk liquid of the chicken eggs.
2. The method according to claim 1, wherein the pathogen is a neocoronavirus and the step of obtaining a pathogen protective antigen gene comprises:
selecting a spike glycoprotein S1 fragment gene sequence containing an RBD structural domain from a genome sequence of the new coronavirus, and synthesizing a corresponding pathogen protective antigen gene by chicken codon optimization.
3. The method according to claim 2, wherein the step of obtaining the recombinant AAV vector vaccine carrying the antigenic protein comprises:
carrying out double enzyme digestion on the core plasmid pAAV-CMV-eGFP by using EcoRI and BamHI, and recovering and obtaining a vector fragment AAV-CMV;
respectively designing amplification primers of antigen genes of spike glycoprotein S1 and RBD, using an S1 gene fragment of a new coronavirus as a template, amplifying to obtain an S1 antigen gene fragment and an RBD antigen gene fragment of which two ends contain EcoRI and BamHI enzyme cutting sites, performing double enzyme cutting recovery by using EcoRI and BamHI, connecting with a vector fragment AAV-CMV, and converting DH5 α competence to obtain a first core plasmid pAAV-CMV-S1 and a second core plasmid pAAV-CMV-RBD;
extracting the first core plasmid pAAV-CMV-S1 and the second core plasmid pAAV-CMV-RBD without endotoxin, and mixing the extracts with two auxiliary plasmids according to a molar ratio of 1: 1: 1, co-transfecting HEK293T cells with the cationic transfection reagent PEI;
collecting the transfected HEK293T cells, repeatedly freezing and thawing for multiple times, collecting supernate, and adding nuclease for incubation;
and after incubation, collecting supernatant again, and removing impurity proteins and impurities to obtain the recombinant AAV vector vaccine rAAV-S1 and rAAV-RBD.
4. The method according to claim 3, wherein the step of immunocapturing the chicken eggs comprises:
selecting healthy hens, inoculating the recombinant AAV vector vaccine rAAV-S1 and the rAAV-RBD to thoracic muscles of the hens at multiple points according to the dose of 1E +11 vg/hen, and collecting hen eggs after immunization;
disinfecting the eggshell of the egg, selecting the eggshell which is seriously polluted in advance to be disinfected separately, washing the eggshell by clear water, soaking the eggshell and the cleaned eggshell into 0.1 percent benzalkonium bromide water solution with the water temperature of 42 ℃ to disinfect for 15min, then soaking the eggshell and the cleaned eggshell into 90-95 ℃ water bath to disinfect for 5 seconds, taking out the eggshell quickly, airing the eggshell, and storing the eggshell at the temperature of 4 ℃.
5. The method according to claim 1, wherein the yolk antibody is obtained by ammonium sulfate precipitation, comprising the following steps:
diluting the yolk with deionized water by 7 times, adjusting the pH value to 5.2 with 3m L HCl solution with the concentration of 0.1 mol/L, standing for 6h at 4 ℃, centrifuging for 30min at 12000r/min, and filtering to obtain a supernatant;
3 parts of 2m L supernatant are respectively taken, saturated ammonium sulfate solution is added until the final concentration is 40 percent, 60 percent and 80 percent respectively, after overnight at the temperature of 4 ℃, the mixture is centrifuged at 5000r/min for 25min, the supernatant is discarded, and the precipitate is re-dissolved with 2m L pure water respectively.
6. The method according to claim 1, wherein the yolk antibody is obtained by polyethylene glycol precipitation, comprising the steps of:
adding 20m L Tris-HC L buffer solution into the yolk, wherein the concentration of the Tris-HC L buffer solution is 0.1 mol/L, the pH value is 7.6, adding PEG after uniformly mixing until the concentration W/V is 3.5%, fully mixing, and centrifuging at 8000r/min for 25 min;
adding PEG into the supernatant until the final concentration W/V is 12%, mixing thoroughly, centrifuging at 12000r/min for 10min, and discarding the supernatant;
the precipitate was redissolved in 10m L of 1 × PBS, PEG was added to a final concentration of 12% W/V, mixed well, centrifuged at 12000r/min for 10min, the supernatant was discarded, and the precipitate was redissolved in 2m L of PBS.
7. The method according to claim 1, wherein the yolk antibody is obtained by cold ethanol precipitation, comprising the steps of:
diluting the yolk with deionized water by 7 times, adjusting the pH value to 5.2 with 3m L HCl solution with the concentration of 0.1 mol/L, standing for 6h at 4 ℃, centrifuging for 30min at 12000r/min, and filtering to obtain a supernatant;
adding 95% ethanol to a final concentration of V/V to 60%, stirring at 4 deg.C for 30min, and centrifuging at 4 deg.C at 10000r/min for 30min to obtain precipitate;
dissolving the precipitate in 10m L NaCl solution with concentration of 0.028 mol/L, filtering at room temperature to remove fat, adding 95% ethanol into the filtrate to make the final concentration V/V to 30%, mixing well, centrifuging at 4 deg.C at 10000r/min for 30min, and dissolving the precipitate in 2m L PBS solution.
8. The method according to claim 1, wherein the yolk antibody is obtained by a water dilution method, comprising the following steps:
diluting the yolk with deionized pure water, adjusting pH to 7.6, stirring at 4 deg.C for 4 hr, centrifuging, discarding precipitate, filtering the supernatant with 200KDa filter membrane, and concentrating to 2ml with 150KDa protein ultrafiltration tube.
9. An anti-pathogenic hyperimmune yolk antibody produced based on an AAV vector vaccine, produced according to the method of any one of claims 1-8.
10. An anti-pathogenic hyperimmune yolk antibody spray formulation prepared based on an AAV vector vaccine, prepared from the anti-pathogenic hyperimmune yolk antibody of claim 9.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094341A (en) * | 2020-09-10 | 2020-12-18 | 华瑞同康生物技术（深圳）有限公司 | IgY neutralizing antibody for resisting novel coronavirus, and preparation method, preparation and application thereof |
| CN112175074A (en) * | 2020-10-09 | 2021-01-05 | 梁永明 | Industrial application of gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies |
| CN112608382A (en) * | 2020-12-28 | 2021-04-06 | 哈药集团生物疫苗有限公司 | Duck reovirus disease and duck viral hepatitis bigeminal egg yolk antibody and preparation method thereof |
| CN112921007A (en) * | 2021-02-05 | 2021-06-08 | 华中农业大学 | Recombinant adeno-associated virus for preventing cat from infecting new coronavirus, construction method and application thereof |
| CN114470198A (en) * | 2020-10-26 | 2022-05-13 | 上海博满生物科技有限公司 | A spray containing IgY and yolk neutralizing antibody of SARS-CoV-2 for preventing and treating new coronary pneumonia |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556113A (en) * | 2004-01-07 | 2004-12-22 | 珠海百奥生物技术有限公司 | Yolk antibody of anti SARS coronavirus and its preparation method and liquid preparation |
| CN105906711A (en) * | 2016-05-03 | 2016-08-31 | 中农威特生物科技股份有限公司 | Preparation method, product and application of yolk antibody for resisting O-type and A-type foot-and-mouth disease viruses |
| CN109929032A (en) * | 2019-02-26 | 2019-06-25 | 青岛今墨堂生物技术有限公司 | A kind of preparation method of anti-hand-foot-and-mouth-disease chicken yolk antibody |
-
2020
- 2020-03-31 CN CN202010244468.0A patent/CN111411118A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556113A (en) * | 2004-01-07 | 2004-12-22 | 珠海百奥生物技术有限公司 | Yolk antibody of anti SARS coronavirus and its preparation method and liquid preparation |
| CN105906711A (en) * | 2016-05-03 | 2016-08-31 | 中农威特生物科技股份有限公司 | Preparation method, product and application of yolk antibody for resisting O-type and A-type foot-and-mouth disease viruses |
| CN109929032A (en) * | 2019-02-26 | 2019-06-25 | 青岛今墨堂生物技术有限公司 | A kind of preparation method of anti-hand-foot-and-mouth-disease chicken yolk antibody |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094341A (en) * | 2020-09-10 | 2020-12-18 | 华瑞同康生物技术（深圳）有限公司 | IgY neutralizing antibody for resisting novel coronavirus, and preparation method, preparation and application thereof |
| CN112094341B (en) * | 2020-09-10 | 2021-05-18 | 华瑞同康生物技术（深圳）有限公司 | IgY neutralizing antibody for resisting novel coronavirus, and preparation method, preparation and application thereof |
| CN112175074A (en) * | 2020-10-09 | 2021-01-05 | 梁永明 | Industrial application of gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies |
| CN114470198A (en) * | 2020-10-26 | 2022-05-13 | 上海博满生物科技有限公司 | A spray containing IgY and yolk neutralizing antibody of SARS-CoV-2 for preventing and treating new coronary pneumonia |
| CN114470198B (en) * | 2020-10-26 | 2023-05-05 | 上海博满生物科技有限公司 | IgY spray of SARS-CoV-2 egg yolk neutralization antibody for preventing and treating new coronal pneumonia |
| CN112608382A (en) * | 2020-12-28 | 2021-04-06 | 哈药集团生物疫苗有限公司 | Duck reovirus disease and duck viral hepatitis bigeminal egg yolk antibody and preparation method thereof |
| CN112608382B (en) * | 2020-12-28 | 2023-08-18 | 哈药集团生物疫苗有限公司 | Duplex egg yolk antibody for duck reovirus disease and duck viral hepatitis and preparation method thereof |
| CN112921007A (en) * | 2021-02-05 | 2021-06-08 | 华中农业大学 | Recombinant adeno-associated virus for preventing cat from infecting new coronavirus, construction method and application thereof |
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