阅读说明：本技术 抗非洲猪瘟病毒和cd多克隆小分子抗体及制备方法和应用 (anti-African swine fever virus and CD polyclonal small molecule antibody, preparation method and application ) 是由 包晟 杨荣鉴 王长安 廖列扬 曹天福 龚芹 于 2019-03-11 设计创作，主要内容包括：本发明提供一种抗非洲猪瘟病毒IgY抗体、抗非洲猪瘟病毒和CD复合IgY抗体、抗非洲猪瘟病毒和CD多克隆小分子抗体以及复合抗体优化组合应用,针对非洲猪瘟病毒以猪免疫细胞为靶细胞,破坏宿主的免疫组织,使疫苗失效的难题；把导致“免疫抑制”和“免疫逃逸”的抗原蛋白以及在病毒扩散中起重要作用的抗原蛋白作为靶抗原,采用复合抗体通过体液免疫和细胞免疫以及ADCC作用抑灭这些抗原蛋白；既突破“免疫抑制”和“免疫逃逸”两大瓶颈,又保护并激活免疫细胞,清除非洲猪瘟病毒和受感染细胞。这种复合抗体可制成生物药或消毒剂用于预防和治疗非洲猪瘟,也可作为饲料或饲料添加剂或口服剂成份,阻断非洲猪瘟病毒经由口腔和消化道的传播。(The invention provides an anti-African swine fever virus IgY antibody, an anti-African swine fever virus and CD composite IgY antibody, an anti-African swine fever virus and CD polyclonal small molecule antibody and composite antibody optimized combination application, aiming at the problem that the African swine fever virus takes a swine immune cell as a target cell, destroys the immune tissue of a host and makes a vaccine ineffective; antigen proteins causing "immunosuppression" and "immune escape" and antigen proteins playing an important role in virus spreading are used as target antigens, and the antigen proteins are suppressed by humoral immunity, cellular immunity and ADCC action by using a composite antibody; not only breaks through two bottlenecks of 'immunosuppression' and 'immune escape', but also protects and activates immune cells and eliminates African swine fever virus and infected cells. The compound antibody can be prepared into a biological medicine or a disinfectant for preventing and treating African swine fever, and also can be used as a feed or a feed additive or an oral agent component for blocking the transmission of the African swine fever virus through the oral cavity and the digestive tract.)

1. A preparation method of an anti-African swine fever virus antibody, wherein the anti-African swine fever virus antibody is an anti-African swine fever virus IgY antibody and an anti-African swine fever virus and CD composite IgY antibody, and is characterized by comprising the following steps:

s1, preparing antigen: adding Freund's adjuvant into one or more of African swine fever virus antigen recombinant protein, recombinant virus expressing African swine fever virus antigen protein and pig CD recombinant protein, placing in a high-speed homogenizer, and homogenizing at high speed to obtain recombinant protein monovalent antigen, recombinant protein bivalent antigen, recombinant protein composite bivalent antigen, recombinant virus monovalent antigen, recombinant virus bivalent antigen, recombinant protein multivalent antigen and recombinant virus multivalent antigen for immunization;

s2, preparing immune eggs: respectively immunizing laying hens with the recombinant protein monovalent antigen, the recombinant protein bivalent antigen, the recombinant protein composite bivalent antigen, the recombinant virus monovalent antigen, the recombinant virus bivalent antigen, the recombinant virus composite bivalent antigen, the recombinant protein multivalent antigen and the recombinant virus multivalent antigen obtained in the step S1, injecting the laying hens once every two weeks for 2-5 times, taking immune eggs laid by the laying hens at least 12 days after the last injection, and carrying out coding marking to obtain anti-African swine fever virus IgY immune eggs and anti-African swine fever virus and CD composite IgY immune eggs;

s3, preparing crude extracts of the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody;

s4, preparing a pure product solution of the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody;

s5, preparing pure dry powder of the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody;

s6, preparing an African swine fever virus resisting IgY antibody and African swine fever virus and CD composite IgY antibody nano powder;

s7, preparing the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody liposome liquid crystal microcapsule.

2. The method for preparing the antibody against African swine fever virus according to claim 1, wherein the African swine fever virus antigen recombinant protein is an African swine fever virus representative antigen protein expressed and purified by eukaryotic cells or prokaryotic cells; inserting genes of a representative antigen protein of the African swine fever virus into a genome of a virus vector to obtain the recombinant virus expressing the African swine fever virus antigen protein; the African swine fever virus representative antigen protein comprises at least one of VP73, P54, P72, P30, A238L and CD 2V; the porcine CD recombinant protein is a representative porcine CD protein expressed and purified by eukaryotic cells or prokaryotic cells, and the representative porcine CD protein comprises at least one of porcine CD3 protein, porcine CD2 protein and porcine CD8 protein.

3. The method according to claim 1, wherein the African swine fever virus-resistant antibody is prepared by preparing a recombinant protein monovalent antigen and a recombinant protein complex bivalent antigen from an African swine fever virus antigen recombinant protein, preparing a recombinant virus monovalent antigen and a recombinant virus complex bivalent antigen from a recombinant virus expressing the African swine fever virus antigen protein, preparing a recombinant protein bivalent antigen from the African swine fever virus antigen recombinant protein and a swine CD recombinant protein, preparing a recombinant virus bivalent antigen from the recombinant virus expressing the African swine fever virus antigen protein and the swine CD recombinant protein, and preparing a recombinant protein multivalent antigen and a recombinant virus multivalent antigen from the African swine fever virus antigen recombinant protein, the recombinant virus expressing the African swine fever virus antigen protein and the swine CD recombinant protein.

4. The method for preparing anti-African swine fever virus antibody according to claim 1, wherein in step S3, crude extracts of anti-African swine fever virus IgY antibody and anti-African swine fever virus and CD complex IgY antibody are prepared by pure water extraction, chloroform extraction, cold ethanol precipitation or ammonium sulfate precipitation.

5. A preparation method of a polyclonal small molecule antibody against African swine fever virus and CD is characterized by comprising the following steps:

s8, adjusting the pH of the pure anti-African swine fever virus and CD complex IgY antibody solution obtained by the method of claims 1-4 to 3.0-5.0, and adding catalytic protease; fully stirring and dissolving to generate enzymatic reaction, then carrying out low-temperature high-speed rotation and centrifugation, discarding the precipitate to obtain supernatant, and carrying out ultrafiltration on the supernatant to obtain concentrated solution; and performing gel chromatography on the concentrated solution obtained after ultrafiltration, and dialyzing and concentrating the chromatography collection to obtain the anti-African swine fever virus and CD polyclonal micromolecule antibody.

6. The method for preparing anti-African swine fever virus and CD polyclonal small molecule antibody according to claim 5, further comprising:

the long-acting modification step S7 of polyethylene glycol, dextran or polyamino acid is used to prepare the anti-African swine fever virus and CD polyclonal micromolecule antibody, and the long-acting anti-African swine fever virus and CD polyclonal micromolecule antibody is obtained.

7. The method for preparing anti-African swine fever virus and CD polyclonal small molecule antibody according to claim 5, further comprising:

the natural CPPs or artificially synthesized CPPs are adopted to couple the anti-African swine fever virus and CD polyclonal small molecule antibody to obtain the coupled anti-African swine fever virus and CD polyclonal small molecule antibody with strong capability of permeating cell membranes.

8. An anti-African swine fever virus antibody, which is an anti-African swine fever virus IgY antibody and an anti-African swine fever virus and CD composite IgY antibody, and is characterized in that the anti-African swine fever virus antibody is prepared by the preparation method of claims 1-4.

9. An anti-african swine fever virus and CD polyclonal small molecule antibody, characterized by being prepared by the preparation method of claims 5-7.

10. A composition comprising one of an anti-african swine fever virus IgY antibody and an anti-african swine fever virus and CD complex IgY antibody produced by the process of claims 1-4, one of an anti-african swine fever virus and CD polyclonal small molecule antibody produced by the process of claims 5-7, and at least one other pharmaceutically acceptable component.

11. The composition of claim 10, wherein the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody are added with auxiliary materials or base materials to be prepared into at least one of oral liquid, gel, lotion, spray, nasal drop, pill, tablet, effervescent tablet, capsule, soft capsule, disinfectant, water injection and powder injection, and can be prepared into biological medicines or disinfection products for eliminating African swine fever virus infection and preventing and treating African swine fever.

12. The composition of claim 10, wherein the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody are prepared into at least one of injection water and powder for injection, and can be used for emergency treatment of swine seriously infected by African swine fever virus.

13. An anti-African swine fever virus IgY antibody or an anti-African swine fever virus and CD composite IgY antibody and application of the anti-African swine fever virus and CD polyclonal small molecule antibody in preparing drugs and disinfection products for preventing and eliminating infection of African swine fever viruses to pigs and/or preventing and treating African swine fever, which is characterized in that the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody prepared by the preparation method in the claims 1-4 and the anti-African swine fever virus and CD polyclonal small molecule antibody prepared by the preparation method in the claims 5-7 are used.

14. The use of anti-African swine fever virus IgY antibody or anti-African swine fever virus and CD composite IgY antibody in preparing feed and feed additive is characterized in that the monovalent or composite monovalent anti-African swine fever virus IgY antibody dry powder or crude extract dry powder or antibody purified liquid or crude extract prepared by the preparation method of claims 1-4 is added into dry powder or liquid feed of pigs as powdery or liquid feed ingredient or feed additive.

Technical Field

The invention relates to the technical field of antibody engineering and genetic engineering, in particular to an anti-African swine fever virus IgY antibody, an anti-African swine fever virus and CD composite IgY antibody, an anti-African swine fever virus and CD polyclonal small molecule antibody, and a preparation method and application thereof.

Background

African Swine Fever (ASF) is a kind of animal epidemic disease with high infectivity, high morbidity and high mortality caused by African Swine Fever Virus (ASFV), and has great harm to the swine industry in China and in the world.

The African swine fever virus has strong resistance in tissues and environment, and no treatment measures are available so far. More importantly, the African swine fever virus is an arbovirus with very unique DNA, mainly takes mononuclear phagocytes of pigs as infection target cells, directly and indirectly destroys immune tissues of hosts, can cause serious immunosuppression and makes vaccines ineffective; meanwhile, the African swine fever virus also has a special immune escape mechanism. Therefore, the prevention and control technology of African swine fever becomes a world big problem.

At present, African swine fever has been introduced into our country, and the epidemic rapidly spreads across the country. Because the African swine fever virus can survive for months to years in meat foods such as frozen meat, cured ham, half-cooked meat and swill; therefore, in addition to the great difficulty in controlling such epidemic diseases, it also causes significant socioeconomic problems by diffusion through the food chain.

Therefore, research and development of a drug capable of effectively preventing and treating the African swine fever, preventing the spread and spread of the African swine fever, minimizing the accidents of the virulent infectious diseases, which is the most serious challenge of the global medical and scientific community and the most urgent task of scientists around the world.

Disclosure of Invention

The invention aims to provide an anti-African swine fever virus IgY antibody, an anti-African swine fever virus and CD composite IgY antibody, an anti-African swine fever virus and CD polyclonal small molecule antibody, and a preparation method and application thereof, so as to solve the problem that the prior art cannot resist infection and diffusion of African swine fever.

The scheme for solving the technical problems is to provide a preparation method of an anti-African swine fever virus antibody, wherein the anti-African swine fever virus antibody is an anti-African swine fever virus IgY antibody and an anti-African swine fever virus and CD composite IgY antibody, and the preparation method comprises the following steps of:

s1, preparing antigen: adding Freund's adjuvant into one or more of African swine fever virus antigen recombinant protein, recombinant virus expressing African swine fever virus antigen protein and pig CD recombinant protein, placing in a high-speed homogenizer, and homogenizing at high speed to obtain recombinant protein monovalent antigen, recombinant protein bivalent antigen, recombinant protein composite bivalent antigen, recombinant virus monovalent antigen, recombinant virus bivalent antigen, recombinant protein multivalent antigen and recombinant virus multivalent antigen for immunization;

s2, preparing immune eggs: respectively immunizing laying hens with the recombinant protein monovalent antigen, the recombinant protein bivalent antigen, the recombinant protein composite bivalent antigen, the recombinant virus monovalent antigen, the recombinant virus bivalent antigen, the recombinant virus composite bivalent antigen, the recombinant protein multivalent antigen and the recombinant virus multivalent antigen obtained in the step S1, injecting the laying hens once every two weeks for 2-5 times, taking immune eggs laid by the laying hens at least 12 days after the last injection, and carrying out coding marking to obtain anti-African swine fever virus IgY immune eggs and anti-African swine fever virus and CD composite IgY immune eggs;

s3, preparing crude extracts of the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody;

s4, preparing a pure product solution of the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody;

s5, preparing pure dry powder of the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody;

s6, preparing an African swine fever virus resisting IgY antibody and African swine fever virus and CD composite IgY antibody nano powder;

s7, preparing the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody liposome liquid crystal microcapsule.

In the preparation method of the antibody for resisting the African swine fever virus, the African swine fever virus antigen recombinant protein is a representative antigen protein of the African swine fever virus expressed and purified by eukaryotic cells or prokaryotic cells; inserting genes of a representative antigen protein of the African swine fever virus into a genome of a virus vector to obtain the recombinant virus expressing the African swine fever virus antigen protein; the African swine fever virus representative antigen protein comprises at least one of VP73, P54, P72, P30, A238L and CD 2V; the porcine CD recombinant protein is a representative porcine CD protein expressed and purified by eukaryotic cells or prokaryotic cells, and the representative porcine CD protein comprises at least one of porcine CD3 protein, porcine CD2 protein and porcine CD8 protein.

In the preparation method of the anti-African swine fever virus antibody, the recombinant protein monovalent antigen and the recombinant protein composite bivalent antigen are prepared by the African swine fever virus antigen recombinant protein, the recombinant virus monovalent antigen and the recombinant virus composite bivalent antigen are prepared by the recombinant virus expressing the African swine fever virus antigen protein, the recombinant protein bivalent antigen is prepared by the African swine fever virus antigen recombinant protein and the swine CD recombinant protein, the recombinant virus expressing the African swine fever virus antigen protein and the swine CD recombinant protein are used for preparing the recombinant protein multivalent antigen and the recombinant virus multivalent antigen.

In the preparation method of the antibody against African swine fever virus, in step S3, a pure water extraction method, a chloroform extraction method, a cold ethanol precipitation method or an ammonium sulfate precipitation method is adopted to prepare crude extracts of the antibody against African swine fever virus IgY and the antibody against African swine fever virus and CD composite IgY.

The invention also provides a preparation method of the anti-African swine fever virus and CD polyclonal small molecule antibody, which is characterized by comprising the following steps:

s8, adjusting the pH of the pure anti-African swine fever virus and CD complex IgY antibody solution obtained by the method of claims 1-4 to 3.0-5.0, and adding catalytic protease; fully stirring and dissolving to generate enzymatic reaction, then carrying out low-temperature high-speed rotation and centrifugation, discarding the precipitate to obtain supernatant, and carrying out ultrafiltration on the supernatant to obtain concentrated solution; and performing gel chromatography on the concentrated solution obtained after ultrafiltration, and dialyzing and concentrating the chromatography collection to obtain the anti-African swine fever virus and CD polyclonal micromolecule antibody.

The preparation method of the anti-African swine fever virus and CD polyclonal small molecule antibody further comprises the following steps:

the long-acting modification step S7 of polyethylene glycol, dextran or polyamino acid is used to prepare the anti-African swine fever virus and CD polyclonal micromolecule antibody, and the long-acting anti-African swine fever virus and CD polyclonal micromolecule antibody is obtained.

The preparation method of the anti-African swine fever virus and CD polyclonal small molecule antibody further comprises the following steps:

the natural CPPs or artificially synthesized CPPs are adopted to couple the anti-African swine fever virus and CD polyclonal small molecule antibody to obtain the coupled anti-African swine fever virus and CD polyclonal small molecule antibody with strong capability of permeating cell membranes.

The invention also provides an anti-African swine fever virus antibody, which is an anti-African swine fever virus IgY antibody and an anti-African swine fever virus and CD composite IgY antibody and is prepared by using the preparation method of the anti-African swine fever virus antibody.

The invention also provides a polyclonal small molecule antibody against African swine fever virus and CD, which is prepared by the preparation method of the polyclonal small molecule antibody against African swine fever virus and CD.

The invention also provides a composition, which comprises one of the African swine fever virus-resistant IgY antibody and the African swine fever virus-resistant and CD composite IgY antibody prepared by the preparation method of the African swine fever virus-resistant antibody, the African swine fever virus-resistant and CD polyclonal micromolecule antibody prepared by the preparation method of the African swine fever virus-resistant and CD polyclonal micromolecule antibody and at least one other pharmaceutically acceptable component.

In the composition provided by the invention, the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody are added with auxiliary materials or base materials to be prepared into at least one of oral liquid, gel, lotion, spray, nasal drop, pill, tablet, effervescent tablet, capsule, soft capsule, disinfectant, water injection and powder injection, and can be used for preparing biological medicines and disinfection products for eliminating African swine fever virus infection and preventing and treating African swine fever.

In the composition provided by the invention, the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody are prepared into at least one of injection water injection and powder injection, and can be used for emergency treatment of swine seriously infected by African swine fever virus.

The invention also provides an anti-African swine fever virus IgY antibody or an anti-African swine fever virus and CD composite IgY antibody and application of the anti-African swine fever virus and CD polyclonal small molecule antibody in preparation of drugs and disinfection products for preventing and eliminating infection of African swine fever viruses on pigs and/or preventing and treating African swine fever, the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody prepared by the preparation method of the anti-African swine fever virus antibody, and the anti-African swine fever virus and CD polyclonal small molecule antibody prepared by the preparation method of the anti-African swine fever virus and CD polyclonal small molecule antibody.

The invention also provides an application of the African swine fever virus IgY antibody or the African swine fever virus and CD composite IgY antibody in preparing feed and feed additives, which is characterized in that the dry powder or the dry crude extract powder or the antibody purified liquid or the crude extract liquid of the anti-African swine fever virus IgY antibody which is prepared according to the above claim is added into dry powder or liquid pig feed as dry powder or liquid feed ingredients or feed additives.

The application of the African swine fever virus-resistant IgY antibody or the African swine fever virus-resistant and CD composite IgY antibody, the African swine fever virus-resistant and CD polyclonal small molecule antibody or the nanoliposomal African swine fever virus-resistant and CD composite IgY antibody can bring the following four beneficial effects:

1. the traditional mode of vaccine prevention is changed, and the problem that the existing vaccines tested globally are ineffective is solved: at present, the prevention of the African swine fever by using the vaccine is mainly researched in all countries in the world, but firstly, the African swine fever virus is a rare genetic multiple-variant DNA virus, the gene types are many, the number is large, and the vaccine can only be used for aiming at a plurality of models and is ineffective for most models; secondly, the African swine fever virus is easy to mutate, the development speed of the vaccine cannot follow the virus mutation speed, and the effect of the vaccine is greatly reduced due to the changeability of the virus antigen of the African swine fever virus in the infection and passage processes; thirdly, the vaccine only has a preventive effect and no therapeutic effect, and once the pig is infected with the disease, the vaccine can only frost on the snow. The invention changes the traditional mode of stimulating the pig to generate the antibody in vivo by injecting the vaccine, and adopts a passive immunization mode of directly inputting the high-activity antibody into the pig. The African swine fever virus resisting IgY antibody or the African swine fever virus and CD composite IgY antibody and the African swine fever virus and CD polyclonal small molecule resisting antibody or the nano lipidization African swine fever virus and CD composite IgY antibody can kill the African swine fever virus entering the body through humoral immunity, can block the African swine fever virus adhesion cells, protects the cells from being infected by the virus, and directly plays a role in blocking infection, which is the key point for blocking infection. The African swine fever virus IgY antibody or the African swine fever virus and CD composite IgY antibody and the African swine fever virus and CD composite Fab small molecule antibody can also neutralize the virus newly released locally in infected cell to make it lose diffusion capacity. In addition, the titer of the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody is generally more than 200 times higher than that of the antibody generated by the injection vaccine in vivo (note: the titer of the antibody generated by the injection vaccine in vivo is more than 1:40, namely the antibody is qualified, and the titer of the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody is more than 1:1 ten thousand); thus, the problem that the vaccine-induced antibodies do not have the ability to neutralize african swine fever virus can be solved from another perspective. More importantly, scientific experiments and researches prove that the African swine fever is a fulminating infectious disease transmitted by the fecal oral route, and the occurrence and spread of epidemic situation can be blocked only by cutting off the fecal oral route; direct infusion of highly active antibodies via the oral and digestive tracts becomes more direct and effective in preventing and blocking virulent infectious diseases transmitted by the fecal oral route. In view of the rapid expansion rate of the African swine fever virus worldwide, scientists judge that the feed raw material is one of the main carriers of the African swine fever virus, and the pig feed also becomes an important transmission path of the African swine fever from the infection source to susceptible animals; in order to cut off the source causing the rapid spread of the epidemic situation and solve the problem of headaches in the global pig industry, the crude extract of the anti-African swine fever virus IgY antibody is added into pig feed as a feed additive, for the epidemic area and nearby pig farms, the crude extract of the anti-African swine fever virus and CD composite IgY antibody is preferably used as the feed additive, and the anti-African swine fever virus IgY antibody or the IgY antibody with optimized combination is adopted to inhibit and kill the African swine fever virus mixed in the feed; thereby, the infection of African swine fever virus via oral cavity and digestive tract is eliminated. In addition, the latest research proves that air is also one of the channels for spreading African swine fever virus, so that the invention prepares the anti-African swine fever virus IgY antibody or the optimized combination IgY and the polyclonal small molecular antibody into nasal spray or nasal drop to prevent the African swine fever virus from entering the body from the respiratory tract through the nasal cavity, thereby achieving the purpose of cutting off the air transmission path most directly. In both cases, it is difficult for the vaccine to achieve this effect. Therefore, the anti-African swine fever virus IgY antibody, the anti-African swine fever virus and CD composite IgY antibody, the anti-African swine fever virus and CD polyclonal small molecule antibody or the nano-liposome anti-African swine fever virus and CD composite IgY antibody provided by the invention are better choices for preventing and blocking the African swine fever epidemic situation, and are innovations of the traditional prevention mode.

2. Anti african swine fever virus and CD complex IgY antibodies activate the compromised immune system: the African swine fever virus is a special virus, mainly takes mononuclear phagocytes of pigs as infection target cells, directly and indirectly destroys host immune tissues, causes serious immunosuppression, and causes no effect of the vaccine, the contained apoptosis protein can make the target cells (macrophages and other immune cells) apoptosis, the African swine fever virus does not produce typical neutralizing antibodies no matter natural infection or artificial infection, although the vaccine researched and improved by scientists in various countries in the world recently can induce to produce antibodies with a certain level, but does not have the capability of neutralizing the African swine fever virus, and the purpose of effectively preventing and controlling the African swine fever cannot be achieved, which is the biggest obstacle of vaccine development; in addition, the immune escape mechanism of the African swine fever virus is complex and diverse, the expression of immune regulatory genes can be interfered, and the elimination of host immune cells can be avoided, which is also an important factor influencing the actual effect of the vaccine. The African swine fever virus and CD composite IgY antibody has multiple functions, wherein the first function is to take antigen protein causing 'immunosuppression' and 'immune escape' as target antigen, (1) attack and inhibit P72 protein participating in virus adsorption and cell invasion, prevent the African swine fever virus from adsorbing and invading cells in a pig body, (2) attack and inhibit A238L which is used as an immunosuppressant and has the effect of promoting apoptosis, eliminate immunosuppression and recover immune function, and (3) attack and inhibit P54 protein playing a key role in immune escape, eliminate immune escape, (4) attack and inhibit CD2V protein playing an important role in the process of virus diffusion and lymphocyte injury, prevent the African swine fever virus from diffusing and infecting other healthy cells, protect lymphocytes from being attacked by the African swine fever virus, and strengthen a whole immune system; the second function is to activate T cells to lyse and apoptosis infected cells by using the CD3 antigen protein on the surface of T cells as a target antigen. The anti-African swine fever virus and CD composite IgY antibody inactivates A238L and P54 causing 'immunosuppression' and 'immune escape' and CD2V antigen protein damaging lymphocytes, and activates T cells at the same time; not only eliminating the 'immunosuppression' and 'immune escape', but also protecting and activating immune cells; therefore, the problem that the antibody generated by the vaccine induction does not have the capability of neutralizing the African swine fever virus is solved from another angle.

3. Antibody nanoliposomes increase therapeutic index: the invention prepares the anti-African swine fever virus IgY antibody, the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal micromolecule antibody into microcapsules, nano-microcapsules, micro-emulsions or nano-liposomes, can improve the bioavailability of the antibodies and generate the effect delay function; when the antibody is prepared into an oral preparation or added into feed for pigs to eat, and is used for preventing and treating African swine fever in an oral mode, the nanoliposome dosage form can reduce the damage of gastric and digestive tract enzymes to the biological activity of the antibody. Meanwhile, the prepared microcapsule, nano microcapsule or microemulsion or nano lipidization anti-African swine fever virus IgY antibody, anti-African swine fever virus and CD composite IgY antibody and anti-African swine fever virus and CD polyclonal micromolecule antibody can enhance the ability of entering microcirculation blood vessels and permeating intercellular spaces, so that the antibody can more effectively permeate into the focus part to inhibit and kill the pathogen of the African swine fever virus. In addition, the polyclonal small molecule antibody which is coupled with the cell-penetrating peptide and resists the African swine fever virus and CD can enter cells infected by the African swine fever virus through endocytosis to play a role in cellular immunity; thereby, the therapeutic index is further improved.

4. The comprehensive prevention and treatment of various composite antibodies are optimized and combined: the invention provides a plurality of African swine fever virus resisting IgY antibodies, African swine fever virus resisting and CD composite IgY antibodies and African swine fever virus resisting and CD polyclonal small molecule antibodies which are prepared by immunizing chicken by adopting recombinant proteins or recombinant virus antigens and aiming at six representative antigen proteins, and the VIP products which have univalent and bivalent values, also have multivalence, have the highest effective value and the most complete effect, and also have single or composite univalent and bivalent popular products. In practical application, the method can be optimized and selected according to the type characteristics of the local epidemic African swine fever virus and the disease conditions of epidemic situations and diseased swinery, and the aim of effective prevention and treatment is achieved by adopting the most economic means.

Detailed Description

The invention will now be further illustrated by reference to the following examples:

firstly, preparing an anti-African swine fever virus IgY antibody and an anti-African swine fever virus and CD composite IgY antibody. The African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody are prepared by the following methods:

s1, preparing antigen: adding Freund's adjuvant into one or more of African swine fever virus antigen recombinant protein, recombinant virus expressing African swine fever virus antigen protein and pig CD recombinant protein, placing in a high-speed homogenizer for high-speed homogenization to obtain recombinant protein monovalent antigen, recombinant protein composite bivalent antigen, recombinant virus monovalent antigen, recombinant virus composite bivalent antigen, recombinant protein multivalent antigen and recombinant virus multivalent antigen for immunization;

s2, preparing immune eggs: respectively immunizing laying hens with the recombinant protein monovalent antigen, the recombinant protein composite bivalent antigen, the recombinant virus monovalent antigen, the recombinant virus composite bivalent antigen, the recombinant protein bivalent antigen, the recombinant virus bivalent antigen, the recombinant protein multivalent antigen and the recombinant virus multivalent antigen obtained in the step S1, injecting the laying hens once every two weeks for 2-5 times, taking immune eggs laid by the laying hens at least 12 days after the last injection, and carrying out coding marking to obtain anti-African swine fever virus IgY immune eggs and anti-African swine fever virus and CD composite IgY immune eggs;

s3, preparing crude extracts of the anti-African swine fever virus IgY antibody and the anti-African swine fever virus and CD composite IgY antibody;

s4, preparing a pure product solution of the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody;

s5, preparing an African swine fever virus resisting IgY antibody and African swine fever virus and CD composite IgY antibody nano powder;

s6, preparing the African swine fever virus resisting IgY antibody and the African swine fever virus and CD composite IgY antibody liposome liquid crystal microcapsule.

Specifically, in step S1, the african swine fever virus antigen recombinant protein is an african swine fever virus representative antigen protein expressed and purified by eukaryotic cells or prokaryotic cells; inserting genes of a representative antigen protein of the African swine fever virus into a genome of a virus vector to obtain the recombinant virus expressing the African swine fever virus antigen protein; the African swine fever virus representative antigen protein comprises at least one of VP73, P54, P72, P30, A238L and CD 2V; the porcine CD recombinant protein is a representative porcine CD protein expressed and purified by eukaryotic cells or prokaryotic cells, and the representative porcine CD protein comprises at least one of porcine CD3 protein, porcine CD2 protein and porcine CD8 protein. The method comprises the steps of preparing a recombinant protein monovalent antigen and a recombinant protein composite bivalent antigen by using an African swine fever virus antigen recombinant protein, preparing a recombinant virus monovalent antigen and a recombinant virus composite bivalent antigen by using a recombinant virus expressing the African swine fever virus antigen protein, preparing a recombinant protein bivalent antigen by using the African swine fever virus antigen recombinant protein and a swine CD recombinant protein, preparing a recombinant virus bivalent antigen by using the recombinant virus expressing the African swine fever virus antigen protein and the swine CD recombinant protein, and preparing a recombinant protein multivalent antigen and a recombinant virus multivalent antigen by using the African swine fever virus antigen recombinant protein, the recombinant virus expressing the African swine fever virus antigen protein and the swine CD recombinant protein.

Therefore, step S1 includes:

1. selecting a representative antigenic component:

considering that the protein VP73 of African swine fever virus is a main structural protein and has stable immunogenicity, the P54 protein plays a key role in a mechanism of escaping from a host defense system, the P72 protein is involved in the adsorption and entry of the virus and cells, and an antibody aiming at the P72 can block the binding of the virus and macrophages; the P30 protein is a main structural protein and a strong immunogenic protein, participates in the process of binding the virus with a cell surface receptor, and plays a key role in the virus internalization process; the A238L protein is a bifunctional protein, is a powerful immunosuppressant and has the effect of promoting apoptosis; CD2V is a viral adhesion protein that plays an important role in the spread of viruses and in the damage of lymphocytes. Therefore, the present invention preferably uses these six proteins as the representative antigen component proteins. For the sake of identification, VP73 protein was named protein a, P54 protein was named protein B, P72 protein was named protein C, P30 protein was named protein D, a238L protein was named protein E, and CD2V protein was named protein F.

2. Gene recombination representative antigenic component

(1) Six representative antigen proteins (such as African swine fever virus VP73, P54, P72, P30, A238L, CD2V and the like) (other African swine fever virus proteins can be selected) are expressed in eukaryotic cells or prokaryotic cells and purified to obtain purified representative recombinant proteins which are respectively named as A1, B1, C1, D1, E1 and F1; or six genes of representative antigen proteins such as African swine fever virus VP73, P54, P72, P30, A238L and CD2V (genes of other African swine fever virus proteins can be selected) are inserted into the genome of a virus vector such as papovavirus, adenovirus, herpes virus, poxvirus or RNA virus, recombinant viruses expressing VP73, P54, P72, P30, A238L, CD2V or other proteins of African swine fever virus are constructed and purified, and the purified representative recombinant viruses are obtained and named as A2, B2, C2, D2, E2 and F2 respectively.

(2) The recombinant protein of porcine CD3 or other porcine CD recombinant proteins (such as CD2 and CD8) is expressed by using Escherichia coli, and the recombinant protein of porcine CD3 or other porcine CD recombinant proteins (such as CD2 and CD8) is purified, so that the CD protein is named as G protein for identification.

3. Production of antigens

(1) Making monovalent antigens and composite monovalent antigens

VP73 single recombinant protein or VP73 recombinant protein in six representative recombinant proteins is uniformly mixed with other 5 recombinant proteins or one recombinant protein is uniformly mixed with other 5 recombinant proteins, adjuvants such as Freund's adjuvant are added, the mass ratio of the antigen components of the recombinant proteins to the Freund's adjuvant is (1-10): (10-1), and the recombinant proteins and the composite monovalent antigen are prepared by placing the recombinant proteins and the composite monovalent antigen in a high-speed homogenizer for high-speed homogenization. Respectively A1, (A1+ B1+ C1+ D1+ E1+ F1).

VP73 single recombinant virus or VP73 recombinant virus in six representative recombinant viruses is uniformly mixed with other 5 recombinant viruses or one recombinant virus is uniformly mixed with other 5 recombinant viruses, adjuvants such as Freund's adjuvant are added, the mass ratio of the antigen components of the recombinant viruses to the Freund's adjuvant is (1-10): (10-1), and the recombinant viruses and the composite monovalent antigen are prepared by placing the recombinant viruses and the composite monovalent antigen in a high-speed homogenizer for high-speed homogenization. Respectively A2, (A2+ B2+ C2+ D2+ E2+ F2).

(2) Production of bivalent antigen

One of the six representative recombinant proteins is uniformly mixed with one of the porcine CD3 recombinant protein or other porcine CD (such as CD2 and CD8) recombinant proteins, and then adjuvants such as Freund's adjuvant are added, the mass ratio of the antigen components of the recombinant proteins to the Freund's adjuvant is (1-10) to (10-1), and the recombinant proteins and the recombinant viruses are placed in a high-speed homogenizer for high-speed homogenization to prepare the recombinant protein bivalent antigen or the recombinant virus bivalent antigen. Respectively, A1G, B1G, C1G, D1G, E1G, F1G. A1G can be combined with other 1-5 bivalent antigens to prepare recombinant protein composite bivalent antigen.

One of six representative recombinant viruses is uniformly mixed with one of porcine CD3 recombinant protein or other porcine CD (such as CD2 and CD8) recombinant protein, an adjuvant is added, the mass ratio of antigen components to Freund's adjuvant is (1-10): (10-1), and the mixture is placed in a high-speed homogenizer for high-speed homogenization to prepare the recombinant virus bivalent antigen. Respectively A2G, B2G, C2G, D2G, E2G, F2G. A2G can be combined with other 1-5 bivalent antigens to prepare recombinant virus composite bivalent antigen.

(3) Making multivalent antigens

Mixing one of the six representative recombinant proteins with any 1-5 of the other representative antigenic proteins, or mixing one of the six representative recombinant viruses to be expressed with 1-5 of the other representative recombinant viruses; then mixing with one of the porcine CD3 recombinant protein or other porcine CD (such as CD2 and CD8) recombinant protein uniformly, adding adjuvant, the mass ratio of antigen component to Freund's adjuvant is (1-10): (10-1), placing in a high-speed homogenizer, and homogenizing at high speed to obtain various combined recombinant protein multivalent antigens or recombinant virus multivalent antigens. One of the combinations may be: A1B1G, A1B1C1G, A1B1C1D1G, A1B1C1D1E1G, A1B1C1D1E1F1G, another combination can be: A2B2G, A2B2C2G, A2B2C2D2G, A2B2C2D2E2G, A2B2C2D2E2F2G, and so on.

Specifically, in step S2, the prepared (1) recombinant protein monovalent antigen and complex bivalent antigen or recombinant virus monovalent antigen and complex bivalent antigen, (2) recombinant protein bivalent antigen or recombinant virus bivalent antigen, (3) multiple combined recombinant protein multivalent antigens or recombinant virus multivalent antigens are used to immunize the laying hens respectively; injecting the laying hens once every two weeks for 2-5 times, taking the immune eggs laid by the laying hens at least 12 days after the last injection, and carrying out coding marking to obtain the multiple combined anti-African swine fever virus and CD composite IgY antibody immune eggs. The resulting immunized eggs can be labeled as: a1 immune egg and A2 immune egg, (A1+ B1+ C1+ D1+ E1+ F1) immune egg, (A2+ B2+ C2+ D2+ E2+ F2) immune egg and A1G immune egg, B1G immune egg, C1G immune egg, D1G immune egg, E1G immune egg, F1G immune egg, A2G immune egg, B2G immune egg, C2G immune egg, D2G immune egg, E2G immune egg, F2G immune egg; and so on.

Specifically, in step S3, crude extracts of the african swine fever virus-resistant IgY antibody and the african swine fever virus-resistant CD complex IgY antibody are prepared by pure water extraction, chloroform extraction, cold ethanol precipitation or ammonium sulfate precipitation. The pure water extraction method is taken as an example to illustrate: the method specifically comprises the following steps: washing the prepared African swine fever virus resisting IgY immune eggs or African swine fever virus and CD composite IgY immune eggs with running water, scrubbing and sterilizing with alcohol, breaking the African swine fever virus resisting IgY and CD composite IgY immune eggs with a egg breaker, filtering out egg white by using an egg yolk sieve, leaving egg yolk, and stirring uniformly; adding distilled water 3-8 times of the yolk liquid volume, diluting, mixing, and adjusting pH to 5.5-6.5 with 1.0N HCI solution; further fully and uniformly stirring the diluent with the adjusted pH value, then cooling the diluent to 2-6 ℃, and standing for 12-24 hours; centrifuging the diluted solution at high speed; taking supernatant obtained by separation, and placing the supernatant into an ultrafilter for ultrafiltration and concentration by 10-20 times; then adding sodium alginate solution with the concentration of 1.0-3.0%, slowly adding the sodium alginate solution until the final concentration is 0.1-0.2%, and stirring until turbidity appears; adding 1.0-3.0% CaCl2 solution to final concentration of 0.1-0.2%, stirring, and standing at 3-4 deg.C for 8-12 hr; centrifuging at high speed and taking supernatant to obtain crude extract of anti-African swine fever virus IgY antibody or anti-African swine fever virus and CD composite IgY antibody.

Specifically, in step S4, the crude extract of the prepared african swine fever virus-resistant IgY antibody or crude extract of the african swine fever virus-resistant CD complex IgY antibody is dissolved in M PB phosphate buffer solution with ph of 7.0 and 0.01mol/L, and then the M PB phosphate buffer solution is sequentially subjected to ion exchange column chromatography and gel column chromatography, so as to obtain a pure solution of the african swine fever virus-resistant IgY antibody or the african swine fever virus-resistant CD complex IgY antibody.

Specifically, in step S5, the prepared pure solution of african swine fever virus-resistant IgY antibody or the pure solution of african swine fever virus-resistant CD complex IgY antibody is subjected to freeze drying or intermediate-low temperature spray drying or fluidized bed drying and other drying methods that do not affect the activity of the antibody to prepare the anti-african swine fever virus IgY antibody or the pure dry powder of anti-african swine fever virus-resistant CD complex IgY antibody.

Specifically, in step S6, the prepared African swine fever virus-resistant IgY antibody pure dry powder or African swine fever virus-resistant and CD composite IgY antibody pure dry powder is added into an ultrafine pulverizer to be ground and pulverized, and is processed into ultrafine particles with the size of 1-100nm and the particle size of more than or equal to 15000 meshes, so as to prepare the African swine fever virus-resistant IgY antibody nano powder or the African swine fever virus-resistant and CD composite IgY antibody nano powder.

Specifically, in step S7, lecithin and cholesterol are dissolved in ethyl ether, then the anti-african swine fever virus IgY antibody pure product or the anti-african swine fever virus and CD composite IgY antibody pure product is added with 4mmol/L Phosphate Buffer Solution (PBS) to prepare IgY antibody solution, the solution is ultrasonically treated for 2min (0.5 min per treatment, intermittent 0.5min), the solution is immediately subjected to reduced pressure rotary evaporation in a water bath until the solution is gelatinous, the gel is subjected to phase inversion by vortex oscillation, then the ether is continuously evaporated to the end, further ultracentrifugation (35000r/min, 30min) is performed to separate and remove the uncontained IgY antibody, the precipitate is washed twice with water and centrifuged to obtain the precipitate, and the precipitate is diluted with 10mmol/L PBS to obtain the anti-african swine fever virus IgY antibody liposome liquid crystal microcapsule or the anti-african swine fever virus and CD composite IgY antibody liposome liquid crystal microcapsule.

The prepared African swine fever virus resisting IgY antibody liposome liquid crystal microcapsule or African swine fever virus resisting and CD composite IgY antibody liposome liquid crystal microcapsule is subjected to freeze drying or medium-low temperature spray drying or fluidized bed drying and other drying modes without influencing the activity of the antibody to remove water, and then the nano liposome African swine fever virus resisting IgY antibody powder or the nano liposome African swine fever virus resisting and CD composite IgY antibody powder is prepared.

The crude extract, the pure solution, the pure dry powder, the nano powder and the nano liposome powder of the African swine fever virus-resistant IgY antibody or the African swine fever virus-resistant and CD composite IgY antibody prepared by the method are respectively marked as an A1-IgY antibody, a B1-IgY antibody, a C1-IgY antibody, a D1-IgY antibody, an E1-IgY antibody, a F1-IgY antibody or A1G-IgY antibody, a B1G-IgY antibody, a C1G-IgY antibody, a D1G-IgY antibody, an E1G-IgY antibody and a F1G-IgY antibody according to different antigens of immunity; and so on.

Second, prepare anti African swine fever virus and CD polyclonal small molecular antibody

Adjusting the pH of the prepared African swine fever virus and CD composite IgY antibody pure product solution to 3.0-5.0, and adding catalytic protease; the mass ratio of the catalytic protease is 0.01-0.1%; fully stirring and dissolving to generate enzymatic reaction, then carrying out low-temperature high-speed rotation and centrifugation, discarding the precipitate to obtain supernatant, and carrying out ultrafiltration on the supernatant to obtain concentrated solution; and performing gel chromatography on the concentrated solution obtained after ultrafiltration, and dialyzing and concentrating the chromatography collection to obtain the anti-African swine fever virus and CD polyclonal micromolecule antibody.

The prepared anti-African swine fever virus and CD polyclonal small molecule antibodies are respectively marked as A1G-small molecule antibody, B1G-small molecule antibody, C1G-small molecule antibody, D1G-small molecule antibody, E1G-small molecule antibody and F1G-small molecule antibody according to the corresponding anti-African swine fever virus and CD composite IgY antibody; and so on.

Thirdly, long-acting treatment of anti-African swine fever virus and CD polyclonal small molecule antibody

The long-acting anti-African swine fever virus and CD polyclonal micromolecule antibody is obtained by long-acting modification of the obtained anti-African swine fever virus and CD polyclonal micromolecule antibody by utilizing polyethylene glycol, dextran or polyamino acid.

The invention is described by taking long-acting modification by using polyethylene glycol as an example, but the method is not limited to the method in practical application, and methods such as a chloroform extraction method, a cold ethanol precipitation method, an ammonium sulfate precipitation method and the like can be adopted. The method for long-acting modification by adopting polyethylene glycol comprises the following steps:

adjusting the pH value of a boric acid buffer solution to 6.5-10.0, adding the boric acid buffer solution after the pH value is adjusted into the African swine fever virus and CD composite Fab small molecular antibody to ensure that the concentration of the African swine fever virus and CD composite Fab small molecular antibody is 0.1-0.5mg/mL, adding polyethylene glycol for reaction, wherein the molar ratio of the African swine fever virus and CD composite Fab small molecular antibody to the polyethylene glycol is 1/10-1/30, the reaction temperature is 15-30 ℃, and the reaction time is controlled to be 1-3 h.

Fourth, anti African swine fever virus and CD polyclonal small molecule antibody coupling cell-penetrating peptide

The method comprises the following steps of coupling anti-African swine fever virus and CD polyclonal micromolecule antibodies by using natural CPPs or artificially synthesized CPPs to obtain the coupled anti-African swine fever virus and CD polyclonal micromolecule antibodies with strong capability of permeating cell membranes, wherein one coupling method comprises the following steps:

taking 1-3 parts of powdery African swine fever virus and CD polyclonal small-molecule antibody dry powder, 1-3 parts of CPPs-EGFP1-3 parts of BSA-NS protein microspheres and 5-10 parts of BSA-NS protein microspheres by mass, respectively dissolving in PBS (pH7.5 and 0.01 mol/L) to obtain an African swine fever virus and CD polyclonal small-molecule antibody solution with the concentration of 0.5-1.5g/L, a CPPs-EGFP solution with the concentration of 0.5-1.5g/L and a protein microsphere solution with the concentration of 2.5-5g/L, respectively slowly adding 10-25mol/L ethanol solution according to the volume ratio of 1: 10-1: 30, stirring and reacting at room temperature for 10-30 minutes, respectively adding the reaction mixture of the African swine fever virus and CD polyclonal small-molecule antibody and the reaction mixture of the CPPs-EGFP into a centrifugal tube, respectively taking acetate buffer solution with pH4.5 and 0.01mol/L and PBS with pH7.5 and 0.01mol/L as washing liquid, and centrifugally washing at 4 ℃; adding the BSA-NS protein microsphere reaction mixture into an ultrafiltration centrifugal tube, centrifugally washing with PBS (phosphate buffer solution) with pH of 7.5 and 0.01mol/L at 4 ℃ to remove residual SPDP, respectively obtaining the SPDP derivatives of anti-African swine fever virus and CD polyclonal small molecular antibodies Fab-PDP, CPPs-EGFP-PDP and BSA-NS-PDP, and storing at 4 ℃ for later use; adding a proper amount of DTT into the obtained anti-African swine fever virus and CD polyclonal small molecular antibody Fab-PDP acetate solution to ensure that the final concentration of DTT in a reaction system is 50mmol/L, slightly stirring for 30 minutes at room temperature, adding into an ultrafiltration centrifugal tube, centrifugally washing at 4 ℃ by taking PBS (phosphate buffer solution) with pH7.5 and 0.01mol/L as a washing solution, removing residual DTT to obtain an anti-African swine fever virus and CD polyclonal small molecular antibody Fab-PDP-SH solution, immediately mixing with CPPs-EG-FP-PDP and BSA-NS-PDP solutions, stirring for 15-20 hours at 4 ℃, centrifugally washing by PBS, precipitating, and finally obtaining the CPEG-Ps-FP-BSA-NS-anti-African swine fever virus and CD polyclonal small molecular antibody Fab protein conjugate.

In practical applications, the above A1-IgY antibody, B1-IgY antibody, C1-IgY antibody, D1-IgY antibody, E1-IgY antibody, F1-IgY antibody or A1G-IgY antibody, B1G-IgY antibody, C1G-IgY antibody, D1G-IgY antibody, E1G-IgY antibody, F1G-IgY antibody or A2G-IgY antibody, B2G-IgY antibody, C2G-IgY antibody, D2G-IgY antibody, E2G-IgY antibody, F2G-Y antibody can be arbitrarily combined into a monovalent, bivalent or multivalent anti-African swine fever virus and CD composite IgY antibody composition. Wherein six antibodies such as a collection A1-IgY antibody, B1-IgY antibody, C1-IgY antibody, D1-IgY antibody, E1-IgY antibody, F1-IgY antibody, etc., or six antibodies (collectively referred to as "six-monoclonal antibodies") such as a collection A2-IgY antibody, B2-IgY antibody, C2-IgY antibody, D2-IgY antibody, E2-IgY antibody, F2-IgY antibody, etc., as well as six antibodies such as a collection A1G-IgY antibody, B1G-IgY antibody "), C1G-IgY antibody, D1G-IgY antibody, E1G-IgY antibody, F1G-IgY antibody, etc., or six antibodies such as a collection A2G-IgY antibody, B2-IgY antibody, C2-G-IgY antibody, D2-G-IgY antibody, six antibodies (collectively referred to as" six-monoclonal antibodies "(collectively referred to as" six-IgY antibodies ") against six representative antigens, the antibody composition mixed according to a certain proportion has the highest titer and the most complete efficacy, and is the most preferable item in practical application. As the antibody component added into the feed, an economic product which only comprises an A1-IgY antibody or an A2-IgY antibody or a set of A1-IgY antibody, B1-IgY antibody, C1-IgY antibody, D1-IgY antibody, E1-IgY antibody, F1-IgY antibody and a set of A2-IgY antibody, B2-IgY antibody, C2-IgY antibody, D2-IgY antibody, E2-IgY antibody and F2-IgY antibody can be selected for saving the cost.

anti-African swine fever virus and CD polyclonal small molecule antibodies were also combined in the above manner. In practical application, in order to improve the curative effect, the anti-African swine fever virus and CD composite IgY antibody is preferably mixed with the corresponding anti-African swine fever virus and CD polyclonal small molecule antibody according to a certain proportion and then is used in combination, so that the characteristic that the polyclonal small molecule antibody easily permeates a mucous membrane layer is utilized, and the special advantage that the polyclonal small molecule antibody coupled with the cell-penetrating peptide can enter infected cells to generate the effect of 'cellular immunity' is particularly exerted.

The invention also provides application of the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody in preparation of drugs and disinfection products for preventing and eliminating infection of African swine fever virus to pigs and/or preventing and treating African swine fever.

The invention also provides an application of the African swine fever virus resisting IgY antibody or the African swine fever virus resisting and CD composite IgY antibody in preventing African swine fever virus in preparation of biologically safe feed and feed additives. Because the pig feed is an important transmission path of the African swine fever from an infection source to susceptible animals, the African swine fever virus can survive in feed raw materials for more than 30 days; therefore, the monovalent, bivalent, polyvalent or compound monovalent, bivalent, polyvalent anti-African swine fever virus IgY antibody dry powder or crude extract dry powder thereof can be added into pig feed to produce feed and feed additive for preventing African swine fever.

In addition, the invention also provides a composition which comprises the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody, the anti-African swine fever virus and CD polyclonal small molecule antibody and pharmaceutically acceptable auxiliary materials. The composition can be various pharmaceutical preparations prepared from the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody, the anti-African swine fever virus and CD polyclonal small molecule antibody and auxiliary materials, and can also be prepared animal protection products, disinfection products and the like.

The anti-African swine fever virus IgY antibody or anti-African swine fever virus and CD composite IgY antibody and anti-African swine fever virus and CD polyclonal small molecule antibody or nano-liposome anti-African swine fever virus and CD composite IgY antibody can be prepared into various stable preparations, including but not limited to the following preparations:

preferably, the adjuvant comprises one or more of excipient, filler, solvent, cosolvent, surfactant and capsule adjuvant.

Preferably, the preparation is spray (for disinfection of environment and transportation means and disinfection and purification of pig farm and slaughterhouse), powder (made into pig feed ingredient or feed additive), tablet, oral liquid, pig mouth spray, nasal spray or capsule, etc.

The anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal small molecule antibody or the nano-liposome anti-African swine fever virus and CD composite IgY antibody are prepared into at least one of injection water injection and powder injection. It can be used for the following purposes: the emergency treatment of pigs seriously infected by African swine fever virus.

Or the anti-African swine fever virus IgY antibody or the anti-African swine fever virus and CD composite IgY antibody and the anti-African swine fever virus and CD polyclonal micro-molecule antibody or the nano-liposome anti-African swine fever virus and CD composite IgY antibody are added with auxiliary materials or base materials to be prepared into at least one of oral agents, gels, washing liquids, sprays, nasal drops, pills, tablets, effervescent tablets, capsules, soft capsules, disinfectants, water injections and powder injections, and the biological agents or disinfection products for eliminating the infection of the African swine fever virus and preventing and treating the African swine fever virus can be prepared. Because the pig feed is an important transmission path of the African swine fever from an infection source to susceptible animals, the African swine fever virus can survive in feed raw materials for more than 30 days; therefore, the single-valence univalent, bivalent, multivalent or compound univalent, bivalent, multivalent anti-African swine fever virus IgY antibody dry powder or crude extract dry powder or antibody purification solution or crude extract can be added into dry-powder or liquid pig feed as dry-powder or liquid feed ingredients or feed additives to produce feed and feed additives for preventing African swine fever.

The following is a detailed description of specific examples.