Preparation method of rabbit round small cyst transfer factor

Abstract

The application discloses a preparation method of rabbit round small sac transfer factor, comprising the following steps: 1) mixing water for injection with ground rabbit round small sac, and homogenizing the mixture by a colloid mill to obtain a homogenate; 2) centrifuging the homogenate after cell crushing, performing tangential flow filtration on the harvested supernatant, and collecting the permeate; 3) performing tangential flow filtration on the permeate by using filter membranes with different molecular weight cut-off pore sizes in stages to obtain crude products of rabbit round small sac transfer factor with required molecular weight ranges; 4) mixing at least one crude product with a certain molecular weight range in step 3) to obtain a primary product of rabbit round small sac transfer factor; and 5) adjusting the pH and the osmotic pressure of the primary product of rabbit round small sac transfer factor after virus removal, performing sterilization filtration, and obtaining rabbit round small sac transfer factor. The application adopts the tangential flow filtration in stages, establishes precise and effective separation and concentration technology of rabbit round small sac transfer factor and small molecule purification technology, and can prepare rabbit round small sac transfer factors with various molecular weight ranges according to different technological process flows by selecting filter membranes with different molecular weight cut-off pore sizes.

Description

Technical Field

[0001] This invention belongs to the field of biological products, specifically relating to a method for preparing rabbit round capsule transfer factor. Background Technology

[0002] Transfer factor (TF) is a cytokine released by immune-active T lymphocytes. It is composed of polypeptides and oligonucleotides, with a molecular weight of less than 10 kilodaltons (kDa). It contains no protein, is non-species specific, non-antigenic, and has no toxic side effects. It is a novel immune adjuvant. Tissue ferrugins (TFs) possess broad immunomodulatory activity. On one hand, they exhibit nonspecificity; nonspecific TFs (NTFs) can be isolated from donors without specific antigen stimulation. NTFs can enhance nonspecific immunity in allogeneic or xenogeneic recipient animals by influencing the expression of receptors IFN-γ, IL-2, IL-4, TLR-2, TLR-4, and cAMP. On the other hand, they exhibit specificity; specific TFs (STFs) can be isolated from antigen-stimulated donors. STFs can mediate the activation of recipient macrophages and suppressor T lymphocytes through specific immune information from sensitized lymphocytes, such as a portion of the antigen-specific T lymphocyte receptor β chain, peptide sequences, and the S100A9 protein. This transfers donor-specific cellular immune function to allogeneic or xenogeneic recipient animals, stimulating specific immunity. The intensity of this response is comparable to the immune response produced by secondary exposure to the same pathogen, such as anti-hepatitis B porcine spleen TF. TFs play an important role in the prevention of infectious diseases and immunodeficiency diseases that are currently difficult to control with antibodies or antibiotics.

[0003] TF (transferase) is a heterogeneous mixture of small molecules with a wide molecular weight distribution and biological activity. Some researchers consider TF to be proteins with a length of approximately 44 amino acids, typically with a molecular weight of approximately 3 to 5 kDa. However, TF molecules may have molecular weights outside this range. Furthermore, it is believed that TF comprises three functional parts (inducible moiety, immunosuppressive moiety, and antigen-specific moiety), each containing TF molecules of different molecular weights. Many researchers also believe that TF includes a nucleoside moiety, which can be linked to or separated from the protein molecule. This nucleoside moiety can enhance the ability of TF to induce a secondary immune response in animals and may also be part of either the inducible or inhibitory moiety of TF. Other studies have shown that TF mainly contains at least 581 polypeptides of different molecular weights, ribose, metal elements such as Na, Zn, K, Mg, and Ca, and free amino acids.

[0004] TF (transferase) is derived from animal spleen, placenta, and bursa of Fabricius, among other tissues and organs. Rabbit bursa of Fabricius is a waste product from the processing of infected animals, making it a widely available raw material with broad application and development prospects. Rabbit bursa of Fabricius is considered the mammalian equivalent of the bursa of Fabricius and is an important gut-associated lymphoid tissue in rabbits. Some scholars have proposed that, in vertebrates without a bursa of Fabricius, the equivalent organ may be gut-associated lymphoid tissue. Other studies have found that the differentiation of gut-associated lymphoid tissue does not depend on stimulation by recognizable antigens in the intestinal contents, but rather on certain essential factors. Therefore, it is believed that rabbit gut-associated lymphoid tissue is a central immune organ in mammals, controlling the occurrence and development of humoral immune cell lines. In the cellular components of various regions of rabbit bursa of Fabricius tissue, the number and proportion of T cells are relatively high, while the content of plasma cells in the intraepithelial lymphocytes is abundant. Preliminary findings through histochemical, immunohistochemical, and ultrastructural observations have confirmed the presence of cells of the diffuse neuroendocrine system in rabbit bursa of Fabricius lymphoid tissue, and the number of these cells increases significantly after antigen stimulation. It is evident that the rabbit round sac is a unique organ of rabbits, an important organ that integrates digestion, immunity, and neuroendocrine functions.

[0005] Currently, the methods for preparing rabbit round capsule transfer factor have the following shortcomings:

[0006] (1) TF is a heterogeneous mixture of small molecules with a molecular weight less than 10 kDa and a wide molecular weight distribution, possessing biological activity. It consists of three functional parts (inducing part, immunosuppressive part, and antigen-specific part), each containing TF molecules of different molecular weights. TF molecules of different molecular weights have different biological activities. Therefore, the biological activity, preparation quality, dosage, and clinical application of TF vary depending on the molecular weight of its components. Current TF manufacturing technologies lack effective TF separation and concentration techniques and small molecule purification techniques, and do not have methods for preparing TF molecules of different molecular weights, resulting in the inability to obtain TF molecules with different molecular weight compositions. These limitations restrict the study of the biological activity, mechanism of action, and effective clinical application of TF.

[0007] (2) Due to technological limitations, there may still be rabbit-derived viruses whose characteristics have not been discovered or understood. In the preparation of rabbit round capsule TF, the potential unknown viruses in rabbit round capsules still pose a viral safety risk to rabbit round capsule TF. Therefore, the virus removal / inactivation process is an important and necessary means to ensure the safety of rabbit round capsule TF. Existing TF manufacturing technologies mostly use 5-10kDa ultrafiltration membranes for ultrafiltration or dialysis bags for dialysis to directly obtain the TF product. The process flow does not use a virus removal / inactivation method. Although ultrafiltration membrane ultrafiltration and dialysis bag dialysis have a certain degree of virus retention and removal effect, due to the filter membrane structure characteristics and filtration process nature of ultrafiltration membranes and dialysis bags, ultrafiltration membrane ultrafiltration and dialysis bag dialysis are not safe, reliable and easily verifiable virus removal / inactivation methods, resulting in a significant viral safety risk in the prepared TF. In addition, some TF manufacturing technologies only use one method to remove / inactivate viruses, such as formaldehyde inactivation or β-propiolactone inactivation. Due to the limitations of a single virus removal / inactivation method, there is a possibility that some viruses cannot be completely removed, and the safety of TF cannot be absolutely guaranteed.

[0008] (3) Rabbit round capsules are waste products generated during processing, and their resource utilization has significant practical implications and broad application prospects. Currently, TF raw materials mainly come from animal spleens and placentas, and there are no reports of extracting and separating TF from rabbit round capsules. Although rabbit round capsules have a wide range of raw material sources, they are not an unlimited resource. Existing manufacturing technologies have poor efficiency in utilizing TF raw materials, only using the supernatant after centrifugation of the homogenate for TF preparation, while discarding the centrifuged precipitate. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing rabbit round sac transfer factor without viral safety risks. This method can obtain rabbit round sac transfer factor with a specific molecular weight.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for preparing rabbit round sac transfer factor includes the following steps:

[0012] 1) Select healthy rabbit round sacs, remove the fascia, muscle and fat tissue on the surface of the rabbit round sacs, wash with water for injection, mince with a meat grinder, mix the water for injection with the minced rabbit round sacs, and homogenize with a colloid mill to obtain a homogenate.

[0013] 2) After cell disruption, the homogenate is centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate is mixed with water for injection adjusted to pH 4.0-5.0 with hydrochloric acid, and homogenized using a colloid mill to obtain a homogenate. 0.01%-0.05% pepsin is added, and the mixture is stirred and hydrolyzed at 37°C for 1-5 hours. Cell disruption is further performed, and the mixture is centrifuged to obtain a secondary supernatant. The primary and secondary supernatants are combined, and the harvested supernatant is then tangentially filtered through a 0.22-0.65 μm membrane to collect the permeate.

[0014] 3) Preparation of crude rabbit round capsule transfer factor less than 10kDa: The permeate from step 2) was tangentially filtered using a 10kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 10kDa.

[0015] 4) Preparation of crude rabbit round capsule transfer factor less than 8kDa: The permeate from step 2) was tangentially filtered using an 8kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 8kDa.

[0016] 5) Preparation of crude rabbit round capsule transfer factor less than 5kDa: The permeate from step 2) was tangentially filtered using a 5kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 5kDa.

[0017] 6) Preparation of crude rabbit round capsule transfer factor of 8 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3) is filtered tangentially using an 8 kD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 8 to 10 kDa.

[0018] 7) Preparation of crude rabbit round capsule transfer factor of 5 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3) is filtered tangentially using a 5KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 5 to 10 kDa.

[0019] 8) Preparation of crude rabbit round capsule transfer factor of 3 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3) is filtered tangentially using a 3KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 10 kDa.

[0020] 9) Preparation of crude rabbit round capsule transfer factor of 1 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3) is filtered tangentially using a 1 KD filter membrane, and the filtrate is collected to obtain the crude rabbit round capsule transfer factor of 1 to 10 kDa.

[0021] 10) Preparation of crude rabbit round capsule transfer factor of 5 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 4) is filtered tangentially using a 5 kD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 5 to 8 kDa.

[0022] Alternatively, the crude 5 to 10 kDa rabbit cyst transfer factor from step 7) can be tangentially filtered using an 8 kD filter membrane, and the permeate can be collected to obtain the crude 5 to 8 kDa rabbit cyst transfer factor.

[0023] 11) Preparation of crude rabbit round capsule transfer factor of 3 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 4) is filtered tangentially using a 3KD filter membrane, and the filtrate is collected to obtain the crude rabbit round capsule transfer factor of 3 to 8 kDa.

[0024] Alternatively, the crude 3 to 10 kDa rabbit cyst transfer factor from step 8) can be tangentially filtered using an 8 kD filter membrane, and the permeate can be collected to obtain the crude 3 to 8 kDa rabbit cyst transfer factor.

[0025] 12) Preparation of crude rabbit round capsule transfer factor of 1 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 4) is filtered tangentially using a 1 kD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 8 kDa.

[0026] Alternatively, the crude 1 to 10 kDa rabbit cyst transfer factor from step 9) can be tangentially filtered using an 8 kD filter membrane, and the permeate can be collected to obtain the crude 1 to 8 kDa rabbit cyst transfer factor.

[0027] 13) Preparation of crude rabbit round capsule transfer factor of 3 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 5) is filtered tangentially using a 3KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 5 kDa.

[0028] Alternatively, the crude rabbit cyst transfer factor of 3 to 10 kDa in step 8) or the crude rabbit cyst transfer factor of 3 to 8 kDa in step 11) can be tangentially filtered using a 5 kD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 3 to 5 kDa.

[0029] 14) Preparation of crude rabbit round capsule transfer factor of 1 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 5) is filtered tangentially using a 1 kD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 5 kDa.

[0030] Alternatively, the crude rabbit cyst transfer factor of 1 to 10 kDa in step 9) or the crude rabbit cyst transfer factor of 1 to 8 kDa in step 12) can be tangentially filtered using a 5 kD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 1 to 5 kDa.

[0031] 15) Preparation of crude rabbit cyst transfer factor less than 3kDa: The crude rabbit cyst transfer factor less than 10kDa in step 3), the crude rabbit cyst transfer factor less than 8kDa in step 4), or the crude rabbit cyst transfer factor less than 5kDa in step 5) are subjected to tangential flow filtration using a 3KD filter membrane. The permeate is collected, which is the crude rabbit cyst transfer factor less than 3kDa.

[0032] Alternatively, the permeate from step 2) can be tangentially filtered using a 30-500kDa filter membrane, and the permeate can be collected and then tangentially filtered again using a 3KD filter membrane. The collected permeate is the crude product of rabbit round capsule transfer factor with a value of less than 3kDa.

[0033] 16) Preparation of crude rabbit round capsule transfer factor of 1 to 3 kDa: The crude rabbit round capsule transfer factor of less than 3 kDa in step 15) is filtered tangentially using a 1 kD filter membrane, and the filtrate is collected to obtain the crude rabbit round capsule transfer factor of 1 to 3 kDa.

[0034] Alternatively, the crude rabbit cyst transfer factor of 1 to 10 kDa in step 9), the crude rabbit cyst transfer factor of 1 to 8 kDa in step 12), or the crude rabbit cyst transfer factor of 1 to 5 kDa in step 14) can be tangentially filtered using a 3 KD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 1 to 3 kDa.

[0035] 17) Preparation of crude rabbit cyst transfer factor less than 1 kDa: The crude rabbit cyst transfer factor less than 10 kDa in step 3), the crude rabbit cyst transfer factor less than 8 kDa in step 4), the crude rabbit cyst transfer factor less than 5 kDa in step 5), or the crude rabbit cyst transfer factor less than 3 kDa in step 15) are subjected to tangential flow filtration using a 1 kDa filter membrane. The permeate is collected, which is the crude rabbit cyst transfer factor less than 1 kDa.

[0036] Alternatively, the permeate from step 2) can be tangentially filtered using a 30-300 kDa filter membrane, and the permeate can be collected and then tangentially filtered again using a 1 kDa filter membrane. The collected permeate is the crude rabbit round capsule transfer factor with a value of less than 1 kDa.

[0037] 18) Mix the crude rabbit round capsule transfer factor products with at least one molecular weight range from steps 3) to 17) to obtain the primary rabbit round capsule transfer factor product.

[0038] 19) After removing the virus from the primary rabbit round capsule transfer factor product by the virus removal / inactivation method, adjust the pH to 6.5-7.5 and the osmotic pressure to 280-320 mosm / kg, and sterilize it with a 0.1-0.22μm sterilization filter to obtain rabbit round capsule transfer factor.

[0039] Further, in step 1), if a healthy rabbit is positive for antibodies after being immunized with a vaccine or stimulated by an antigen, then step 19) obtains rabbit round vesicle transfer factor specific to that vaccine or antigen; conversely, if a healthy rabbit is negative for antibodies without being immunized with a vaccine or stimulated by an antigen, then step 19) obtains non-specific rabbit round vesicle transfer factor.

[0040] Furthermore, in step 2), the cell disruption method includes at least one of the following methods: ① repeated freeze-thaw disruption; ② disruption using a high-pressure homogenizer; ③ disruption using an ultrasonic cell disruptor.

[0041] Further, in step 19), the virus removal / inactivation method includes at least two of the following methods: ① low pH incubation method (reaction at 4-25℃ for 2h-1d at pH 2.0-4.0); ② membrane filtration method (filtration using a 15-45nm virus removal filter); ③ β-propiolactone inactivation method (inactivation at 4℃ for 6-24h at a concentration of 0.001%-0.025%, followed by hydrolysis at 37℃ for 2-8h); ④ formaldehyde inactivation method (inactivation at 37℃ for 6-12h at a concentration of 0.01%-0.05%).

[0042] When the volume ratio of rabbit round capsule transfer factor with a molecular weight of 3 to 5 kDa is 40%-70%, it is particularly suitable for improving the specific immune level of animals.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. This invention employs a stepwise tangential flow filtration process to establish a precise and effective separation and concentration technology and small molecule purification technology for rabbit cytosolic granulocyte transfer factor (TF). By selecting filter membranes with different pore sizes and molecular weight cutoffs and following different process flows, crude rabbit cytosolic granulocyte transfer factor (TCF) products with molecular weights less than 10 kDa, less than 8 kDa, less than 5 kDa, 8-10 kDa, 5-10 kDa, 3-10 kDa, 1-10 kDa, and 5-8 kDa can be prepared. Rabbit cyst transfer factor crude products of different molecular weights (kDa, 3-8kDa, 1-8kDa, 3-5kDa, 1-5kDa, less than 3kDa, and less than 1kDa) are obtained by mixing at least one of these crude rabbit cyst transfer factor products in different proportions, subjecting them to virus removal / inactivation treatment, pH and osmotic pressure adjustment, and sterilization.

[0045] Transfer factors with different molecular weights exhibit different biological activities. Compared with transfer factors prepared by existing transfer factor manufacturing technologies, the rabbit round capsule transfer factor prepared in this invention has different proportions of different molecular weight components, including different volume ratios of rabbit round capsule transfer factors less than 10 kDa, less than 8 kDa, less than 5 kDa, 8 to 10 kDa, 5 to 10 kDa, 3 to 10 kDa, 1 to 10 kDa, 5 to 8 kDa, 3 to 8 kDa, 1 to 8 kDa, 3 to 5 kDa, 1 to 5 kDa, less than 3 kDa, 1 to 3 kDa, or less than 1 kDa. The rabbit round capsule transfer factors prepared in this invention with different molecular weight components exhibit different biological activities in key functions such as induction function, immunosuppression function, and antigen-specific function, and also have different dosages and clinical effects, providing a technical basis for the study of the biological activity, mechanism of action, and effective clinical application of TF.

[0046] 2. Rabbit cyclophosphamide (TB) transfer factor crude product is obtained through processes such as tangential flow filtration. However, this crude product may still be contaminated by inadvertently introduced exogenous viruses or potentially unknown viruses. To eliminate any remaining exogenous viruses in the crude product and prevent viruses from entering the finished TB product, it is necessary to treat it with virus removal / inactivation methods. Current transfer factor manufacturing technologies often employ ultrafiltration membranes or dialysis bags to remove viruses, which are unreliable and difficult to verify. Alternatively, they may use a single virus removal / inactivation method (e.g., formaldehyde inactivation or β-propiolactone inactivation). These methods may not completely remove some viruses, failing to guarantee the absolute safety of the TF and resulting in a significant viral safety risk for the prepared TF. This invention validates and evaluates methods for removing / inactivating rabbit round capsule TF. From various methods including pasteurization, dry heat treatment, organic solvent / detergent (S / D) treatment, membrane filtration, low pH incubation, β-propiolactone inactivation, formaldehyde inactivation, diethyleneimine inactivation, octanoic acid treatment, photochemical methods, deep filtration, and chromatography, a method for removing / inactivating the virus with a wide range of inactivations, good efficacy, and proven success, while maintaining the potency and stability of rabbit round capsule TF, was screened and established. Compared with existing transfer factor manufacturing technologies, this invention includes at least two of the following methods for virus removal / inactivation: ① low pH incubation method (reaction at 4-25℃ for 2h-1d at pH 2.0-4.0); ② membrane filtration method (filtration using a 15-45nm virus removal filter); ③ β-propiolactone inactivation method (inactivation at 4℃ for 6-24h at a concentration of 0.001%-0.025%, followed by hydrolysis at 37℃ for 2-8h); ④ formaldehyde inactivation method (inactivation at 37℃ for 6-12h at a concentration of 0.01%-0.05%). This clarifies that in the production of rabbit round capsule TF, two or more virus removal / inactivation methods with different mechanisms must be employed to ensure complete virus inactivation / removal, thereby guaranteeing the safety of rabbit round capsule TF.

[0047] 3. Rabbit round capsules are waste products from the processing of rabbits, and their resource utilization has significant practical importance and broad application prospects. In existing technologies, TF raw materials mainly come from animal spleens and placentas, and there are no reports of extracting and isolating TF from rabbit round capsules. This invention is the first to prepare rabbit round capsule transfer factor without viral safety risks from rabbit round capsules.

[0048] 4. Although rabbit round capsules (TFs) have a wide range of raw material sources, they are not an unlimited resource. Existing manufacturing technologies have poor utilization efficiency for TF raw materials, using only the supernatant from centrifugation of the homogenate for TF preparation, while discarding the centrifuged precipitate. In this invention, the centrifuged precipitate undergoes a secondary extraction: the precipitate is further mixed with water for injection adjusted to pH 4.0-5.0 with hydrochloric acid, homogenized using a colloid mill, and then 0.01%-0.05% pepsin is added. After stirring and hydrolyzing at 37°C for 1-5 hours, cell disruption is further performed, and the supernatant is obtained by centrifugation for TF preparation. This invention uses water for injection adjusted to pH 4.0-5.0 with hydrochloric acid for mixing and homogenizing the centrifuged precipitate, which is beneficial for inhibiting enzyme activity and achieving high-efficiency extraction of rabbit round capsule TF. It also provides a good liquid solvent environment for subsequent activation of pepsin activity, realizing secondary extraction of the centrifuged precipitate and improving the yield of rabbit round capsule TF. Detailed Implementation

[0049] Example 1

[0050] A method for preparing rabbit round sac transfer factor includes the following steps:

[0051] 1) Select round sacs of healthy rabbits, remove the fascia, muscle and fat tissue on the surface of the rabbit round sacs, wash with water for injection, mince with a meat grinder, mix the water for injection with the minced rabbit round sacs, and homogenize with a colloid mill to obtain a homogenate.

[0052] 2) After cell disruption, the homogenate was centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was then mixed with water for injection adjusted to pH 4.0 with hydrochloric acid, homogenized using a colloid mill, and 0.05% pepsin was added. After stirring and hydrolyzing at 37°C for 5 hours, the cells were further disrupted, and centrifuged to obtain a secondary supernatant. The primary and secondary supernatants were combined, and the harvested supernatant was tangentially filtered through a 0.22 μm filter membrane to collect the permeate.

[0053] The cell disruption method is repeated freeze-thaw disruption.

[0054] 3) Preparation of crude rabbit round capsule transfer factor less than 10kDa: The permeate from step 2) was tangentially filtered using a 10kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 10kDa.

[0055] 4) Preparation of crude rabbit round capsule transfer factor less than 5kDa: The permeate from step 2) was tangentially filtered using a 5kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 5kDa.

[0056] 5) Preparation of crude rabbit round capsule transfer factor of 3 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 4) is tangentially filtered using a 3KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 5 kDa.

[0057] 6) Mix the crude rabbit cyst transfer factor products with different molecular weight ranges prepared in steps 3) to 5), and the mixing ratio is: the crude rabbit cyst transfer factor product with a molecular weight of less than 10 kDa accounts for 30% of the volume of the mixed liquid, and the crude rabbit cyst transfer factor product with a molecular weight of 3 to 5 kDa accounts for 70% of the volume of the mixed liquid. This is the initial product of rabbit cyst transfer factor.

[0058] 7) After removing the virus from the initial rabbit round capsule transfer factor product using the following two methods (low pH incubation method: reaction at 25℃ for 1 day at pH 2.0; formaldehyde inactivation method: inactivation at 37℃ for 6 hours at a concentration of 0.01%), the pH was adjusted to 7.5 and the osmotic pressure was adjusted to 320 mosm / kg. The product was then sterilized using a 0.22 μm sterilization filter to obtain the rabbit round capsule transfer factor.

[0059] In step 1), the healthy rabbits tested positive for swine fever virus antibodies after being immunized with the swine fever virus vaccine. Therefore, step 7) yielded swine fever virus-specific rabbit round vesicle transfer factor.

[0060] The product of this embodiment is particularly suitable for improving animal-specific immune levels (the volume ratio of rabbit round capsule transfer factor with a molecular weight of 3 to 5 kDa is between 40% and 70%).

[0061] Example 2

[0062] A method for preparing rabbit round sac transfer factor includes the following steps:

[0063] 1) Select round sacs of healthy rabbits, remove the fascia, muscle and fat tissue on the surface of the rabbit round sacs, wash with water for injection, mince with a meat grinder, mix the water for injection with the minced rabbit round sacs, and homogenize with a colloid mill to obtain a homogenate.

[0064] 2) After cell disruption, the homogenate was centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was then mixed with water for injection (pH adjusted to 5.0 with hydrochloric acid), and homogenized using a colloid mill. A 0.01% pepsin solution was added, and the mixture was stirred and hydrolyzed at 37°C for 1 hour. Cell disruption was further performed, followed by centrifugation to obtain a secondary supernatant. The primary and secondary supernatants were combined. The harvested supernatant was then tangentially filtered through a 0.45 μm membrane, and the permeate was collected.

[0065] The cell disruption methods are as follows: the first cell disruption is performed using a high-pressure homogenizer, and the second cell disruption is performed using an ultrasonic cell disruptor.

[0066] 3) Preparation of crude rabbit round capsule transfer factor less than 10kDa: The permeate from step 2) was tangentially filtered using a 10kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 10kDa.

[0067] 4) Preparation of crude rabbit round capsule transfer factor less than 5kDa: The permeate from step 2) was tangentially filtered using a 5kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 5kDa.

[0068] 5) Preparation of crude rabbit round capsule transfer factor of 8 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3) is tangentially filtered using an 8 kD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 8 to 10 kDa.

[0069] 6) Preparation of crude rabbit round capsule transfer factor of 3 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 4) is tangentially filtered using a 3KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 5 kDa.

[0070] 7) Mix the crude rabbit round capsule transfer factor products with different molecular weights prepared in steps 4) to 6), and the mixing ratio is as follows: the crude rabbit round capsule transfer factor product with a molecular weight of less than 5 kDa accounts for 30% of the volume of the mixed liquid, the crude rabbit round capsule transfer factor product with a molecular weight of 8 to 10 kDa accounts for 30% of the volume of the mixed liquid, and the crude rabbit round capsule transfer factor product with a molecular weight of 3 to 5 kDa accounts for 40% of the volume of the mixed liquid. This is the initial product of rabbit round capsule transfer factor.

[0071] 8) For the initial rabbit round capsule transfer factor product, after virus removal / inactivation methods (the following two methods were used in sequence: β-propiolactone inactivation method: inactivation at 4℃ for 24h at a concentration of 0.025% followed by hydrolysis at 37℃ for 2h; membrane filtration method: filtration using a 45nm virus removal filter), the pH was adjusted to 6.5, the osmotic pressure was adjusted to 282mosm / kg, and sterilization was performed using a 0.1μm sterilization filter to obtain rabbit round capsule transfer factor.

[0072] In step 1), the healthy rabbits tested positive for swine fever virus antibodies after being immunized with the swine fever virus vaccine. Therefore, step 8) yielded swine fever virus-specific rabbit round vesicle transfer factor.

[0073] The product of this embodiment is particularly suitable for improving animal-specific immune levels (the volume ratio of rabbit round capsule transfer factor with a molecular weight of 3 to 5 kDa is between 40% and 70%).

[0074] Example 3

[0075] A method for preparing rabbit round sac transfer factor, comprising the following steps:

[0076] 1) Select round sacs of healthy rabbits, remove the fascia, muscle and fat tissue on the surface of the rabbit round sacs, wash with water for injection, mince with a meat grinder, mix the water for injection with the minced rabbit round sacs, and homogenize with a colloid mill to obtain a homogenate.

[0077] 2) After the homogenate is disrupted by cell disruption (using an ultrasonic cell disruptor), it is centrifuged to obtain the supernatant and precipitate. The collected supernatant is then tangentially filtered through a 0.65 μm membrane, and the permeate is collected.

[0078] 3) Preparation of crude rabbit round capsule transfer factor less than 5kDa: The permeate from step 2) was tangentially filtered using a 5kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 5kDa.

[0079] 4) Preparation of crude rabbit round capsule transfer factor of 3 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 3) is filtered tangentially using a 3KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 5 kDa.

[0080] 5) Preparation of crude rabbit round capsule transfer factor less than 1kDa: The permeate from step 2) was tangentially filtered using a 300kDa filter membrane. The permeate was collected and then tangentially filtered again using a 1KD filter membrane. The collected permeate was the crude rabbit round capsule transfer factor less than 1kDa.

[0081] 6) Mix the crude rabbit round capsule transfer factor products with different molecular weights prepared in steps 4) to 5) in the following proportions: the crude rabbit round capsule transfer factor products with molecular weights of 3 to 5 kDa account for 55% of the volume of the mixed liquid, and the crude rabbit round capsule transfer factor products with molecular weights less than 1 kDa account for 45% of the volume of the mixed liquid. This is the initial product of rabbit round capsule transfer factor.

[0082] 7) For the initial rabbit round capsule transfer factor product, after virus removal / inactivation methods (the following two methods were used in sequence: low pH incubation method: reaction at 21℃ for 1 day at pH 3.0; β-propiolactone inactivation method: inactivation at 4℃ for 6 hours at 0.001% concentration followed by hydrolysis at 37℃ for 8 hours), the pH was adjusted to 7.2, the osmotic pressure was adjusted to 310 mosm / kg, and sterilization was carried out using a 0.22μm sterilization filter to obtain rabbit round capsule transfer factor.

[0083] In step 1), the healthy rabbits tested positive for Newcastle disease virus antibodies after being immunized with chicken Newcastle disease virus vaccine. Therefore, step 7) yielded Newcastle disease virus-specific rabbit round cyst transfer factor.

[0084] The product of this embodiment is particularly suitable for improving animal-specific immune levels (the volume ratio of rabbit round capsule transfer factor with a molecular weight of 3 to 5 kDa is between 40% and 70%).

[0085] The following comparative experiments, using examples 1-3, compare the rabbit round cyst transfer factor prepared in this invention with existing rabbit round cyst transfer factors.

[0086] Comparative Example 1

[0087] A comparative experiment was conducted to examine the specific efficacy of the rabbit cyst transfer factor prepared in Example 1 of this invention with existing rabbit cyst transfer factors in a leukocyte adhesion inhibition assay.

[0088] The existing methods for preparing rabbit round capsule transfer factor used in this experiment are as follows:

[0089] The cytosolic vesicles of healthy rabbits that tested positive for swine fever virus antibodies after immunization with swine fever virus vaccine were crushed, mixed with water for injection, homogenized, and subjected to repeated freeze-thaw cycles before centrifugation. The supernatant was then filtered through a 0.22 μm filter membrane and a 10 kDa filter membrane. The permeate was collected, and the pH was adjusted to 7.2 and the osmotic pressure was adjusted to 290 mosm / kg. After sterilization and filtration, the resulting product was the existing rabbit cytosolic vesicle transfer factor.

[0090] This experiment employed a leukocyte adhesion inhibition assay to detect the specific potency of different rabbit cytomegalovirus transfer factors (CMFs). The leukocyte adhesion inhibition assay is based on the principle that CMFs specific to classical swine fever virus (CSFV) can induce non-sensitized leukocytes to acquire CSFV-specific immune information, thereby transforming them into sensitized leukocytes. Under the influence of CSFV, the adhesion of sensitized leukocytes to glass or plastic surfaces was inhibited. The specific activity of the CMFs was determined by calculating the Non-Adhesion Inhibition Index (NAI) based on the number of non-adhering leukocytes.

[0091] 1. Solution preparation

[0092] 1.1 Hank's solution: Mix 0.3% potassium dihydrogen phosphate solution, 0.76% disodium hydrogen phosphate solution, 2% potassium chloride solution and 20% sodium chloride solution in a ratio of 20:20:20:40, add 1g of glucose, dissolve and mix well, dilute with water to 1000mL, and adjust the pH to 7.2-7.3 with 4% sodium bicarbonate solution (prepare immediately before use).

[0093] 1.2 The separation solution was a lymphocyte separation solution.

[0094] 1.3 2% trypan blue staining solution: Weigh 0.4g of trypan blue and place it in a mortar. Add a small amount of purified water and grind repeatedly. Add purified water to 100mL, centrifuge at 1500r / min for 10min, and take the supernatant, which is the 4% trypan blue aqueous solution. Before use, dilute it 1:1 with 1.8% NaCl solution to obtain the 2% trypan blue staining solution.

[0095] 2. Operation Method

[0096] 2.1 Preparation of Mouse Leukocyte Suspension Healthy mice weighing 22–25 g were selected and euthanized by cervical dislocation. Fresh spleens were aseptically removed, washed with Hank's solution to remove surface blood, fat, and gently crushed. An appropriate amount of Hank's solution was added to suspend the cells. The suspension was filtered through a 100-mesh sieve, centrifuged at 1500 rpm for 3–5 min, and the supernatant was discarded. A small amount of Hank's solution was added and mixed well. This solution was added to a centrifuge tube containing half the filtrate, and centrifuged at 2000 rpm for 20 min. The spleen cells in the middle layer were carefully aspirated and transferred to another centrifuge tube. An appropriate amount of Hank's solution was added for washing, and the mixture was shaken well. The cells were centrifuged at 1500 rpm for 3–5 min, and the supernatant was discarded. The washing process was repeated once. An appropriate amount of Hank's solution was added to the precipitate, mixed well, and then appropriately diluted with Hank's solution. The cells were counted to obtain a final concentration of (1.0 × 10⁻⁶) cells per mL. 7 )~(2.0×10 7 ( ) cells, which is a suspension of mouse leukocytes.

[0097] 2.2 Preparation of the test sample: The rabbit round capsule transfer factor of Example 1 of the present invention and the existing rabbit round capsule transfer factor were prepared into a test sample containing 1.0 mg of polypeptide per 1.0 mL using Hank's solution.

[0098] 2.3 Preparation of virus solution: Take the attenuated swine fever rabbit virus and dilute it appropriately with Hank's solution to make the titer of the attenuated swine fever rabbit virus 50,000 RID / 0.1 mL.

[0099] 2.4 Determination Method

[0100] A 24-well microplate was prepared, with experimental and control groups, and three replicate wells for each group. 150 μl of the test sample and 150 μl of mouse leukocyte suspension were added to each well in the experimental group, while 150 μl of Hanks's solution and 150 μl of mouse leukocyte suspension were added to each well in the control group. The plates were incubated at 37°C and 5% CO2 for sensitization. After 30 min, 150 μl of attenuated classical swine fever virus (CSFV) was added to each well in each group, and the plates were incubated at 37°C and 5% CO2 for another 2 h. The microplate was then shaken for 1 min to remove any unattached mouse leukocytes, and the supernatant from each well was aspirated. 450 μl of Hanks's solution was then added to each well in each group, and the plates were shaken for another 1 min to remove any unattached mouse leukocytes, and the supernatant from each well was aspirated. The supernatants from both wells were combined (as shown in Table 1).

[0101] Table 1. Procedure for Leukocyte Adhesion Inhibition Method

[0102]

[0103] 2.5 count

[0104] Add 50 μl of 2% trypan blue staining solution to the supernatant of each well in each group, mix well, let stand for 4 min, and count. Count the number of all live white blood cells on the 64 large squares of the counting plate.

[0105] 3 Result Calculation

[0106] The result is calculated using the following formula: NAI = (SC) / C × 100%

[0107] In the formula, NAI: Non-adherent leukocyte suppression index;

[0108] S: Average number of non-adherent white blood cells in the experimental group;

[0109] C: Average number of non-adherent white blood cells in the control group.

[0110] 4 Results

[0111] The results of the leukocyte adhesion inhibition assay (as shown in Table 2) indicate that the NAI value of the rabbit cyst transfer factor of Example 1 of the present invention is significantly higher than that of existing rabbit cyst transfer factors, at 51.8% and 36.5%, respectively. These results demonstrate that the leukocyte adhesion inhibition assay of the rabbit cyst transfer factor of Example 1 of the present invention has significantly higher specific efficacy than that of existing rabbit cyst transfer factors.

[0112] Table 2 Results of the leukocyte adhesion inhibition test

[0113]

[0114] Comparative Example 2

[0115] A comparative experiment was conducted on the effect of the rabbit round vesicle transfer factor prepared in Example 2 of this invention on the immune antibodies against classical swine fever vaccine, compared with existing rabbit round vesicle transfer factors.

[0116] The existing methods for preparing rabbit round capsule transfer factor used in this experiment are as follows:

[0117] Rabbit cyst transfer factor was obtained from healthy rabbits that were immunized with swine fever virus vaccine and tested positive for swine fever virus antibodies. The cysts were cleaned, crushed, treated with boiling water bath, extracted with 5% acetic acid solution, centrifuged, and the supernatant was collected. The pH was adjusted to 7.0, centrifuged again, and the supernatant was separated by Sephadex G100 column and filtered with a 10KD filter membrane. The peak value below 10KD was collected by ultraviolet monitoring and is the existing rabbit cyst transfer factor.

[0118] This experiment used the indirect hemagglutination antibody detection method for classical swine fever to detect the effect of rabbit round vesicle transfer factor of Example 2 of the present invention and existing rabbit round vesicle transfer factor on the immune antibodies of classical swine fever vaccine.

[0119] 1. Experimental Materials

[0120] 1.1 Animals: Healthy Duroc × Landrace × Landrace piglets aged 4–5 weeks that are negative for swine fever antibodies.

[0121] 1.2 Reagents: Swine Fever Indirect Hemagglutination Antibody Detection Kit

[0122] 1.3 Developed swine fever live vaccine (cell-derived).

[0123] 2. Experimental Methods

[0124] Twenty-four healthy piglets were randomly divided into four groups of six each. Experimental group 1 received a combined immunization with one dose of classical swine fever (CSF) vaccine diluted in 2.0 ml of rabbit cyst transfer factor (as described in Example 2 of this invention). Experimental group 2 received a combined immunization with one dose of CSF vaccine diluted in 2.0 ml of existing rabbit cyst transfer factor. The control group received one dose of CSF vaccine diluted in 2.0 ml of physiological saline. A blank control group (injected only with physiological saline) was also included. All groups received intramuscular injection in the neck at a dose of 2.0 ml per piglet. Both the experimental and control groups received one dose of CSF vaccine per piglet (as shown in Table 3). Blood samples were collected from each group on days 7, 14, 28, and 56 post-immunization, and serum was separated to detect the titer of CSF indirect hemagglutination antibodies. The data were analyzed using one-way ANOVA and the least significant difference (LSD) test. P < 0.05 was considered statistically significant.

[0125] Table 3 Experimental Design

[0126]

[0127] 3 Results

[0128] The results showed (as shown in Table 4) that on days 7, 14, 28, and 56 after immunization with the classical swine fever live vaccine, the indirect hemagglutination antibody titers (log2) of experimental groups 1 and 2 were significantly higher than those of the control group (P<0.05). The indirect hemagglutination antibody titers (log2) of experimental group 1 were 1.50, 2.33, 2.00, and 1.33 higher than those of the control group, respectively, while those of experimental group 2 were 1.33, 1.33, 1.00, and 1.17 higher than those of the control group, respectively.

[0129] On days 14 and 28 post-immunization, the titer (log2) of classical swine fever indirect hemagglutination antibody in experimental group 1 was significantly higher than that in experimental group 2 (P<0.05), both increasing by 1.00. The results indicate that rabbit sclerotium transfer factor can significantly enhance the immune antibodies against classical swine fever vaccine, and the immunomodulatory effect of the rabbit sclerotium transfer factor in Example 2 of this invention is significantly higher than that of existing rabbit sclerotium transfer factors.

[0130] Table 4. Test Results

[0131]

[0132] Note: Different lowercase superscripts indicate significant differences (P<0.05); the difference is the difference between each experimental group and the control group.

[0133] Comparative Example 3

[0134] A comparative experiment was conducted on the effect of the rabbit round sac transfer factor prepared in Example 3 of this invention on the immune antibodies against Newcastle disease vaccine in chickens, compared with existing rabbit round sac transfer factors.

[0135] The existing methods for preparing rabbit round capsule transfer factor used in this experiment are as follows:

[0136] The cystic vesicles of healthy rabbits that tested positive for Newcastle disease virus antibodies after immunization with Newcastle disease virus vaccine were minced, mixed with water for injection, homogenized, and then centrifuged using an ultrasonic cell disruptor. The supernatant was then filtered through a 0.45 μm filter membrane and a 5 kDa filter membrane. The permeate was collected, and the pH was adjusted to 7.0 and the osmotic pressure was adjusted to 310 mosm / kg. The permeate was then sterilized through a 0.1 μm sterile filter to obtain the existing rabbit cystic vesicle transfer factor.

[0137] This experiment used the chicken Newcastle disease hemagglutination inhibition test to detect the effect of rabbit round vesicle transfer factor of Example 3 of the present invention and existing rabbit round vesicle transfer factor on the immune antibodies of chicken Newcastle disease vaccine.

[0138] 1. Experimental Materials

[0139] 1.1 Animals: 21-28 day old SPF chickens.

[0140] 1.2 Newcastle disease live vaccine for chickens (La Sota strain).

[0141] 1.3 Reagents: Newcastle disease virus hemagglutination inhibition test antigen, Newcastle disease virus hemagglutination inhibition test positive serum (HI antibody titer of 8log2), and SPF chicken serum.

[0142] 1.4 Solution Preparation

[0143] 1.4.1 Argyle's solution: Dissolve 0.420 g sodium chloride, 0.800 g sodium citrate (2H2O), and 2.050 g glucose in distilled water and dilute to 100 ml. After dissolution, adjust the pH to 6.1 with 10% citric acid and autoclave.

[0144] 1.4.2 pH 7.0–7.2, 0.01 mol / L PBS Solution A (0.2 mol / L disodium hydrogen phosphate): 71.64 g disodium hydrogen phosphate (12H2O), 8.50 g sodium chloride, add distilled water to 1000 ml; Solution B (0.2 mol / L sodium dihydrogen phosphate): 31.21 g sodium dihydrogen phosphate (2H2O), 8.50 g sodium chloride, add distilled water to 1000 ml. Measure 72.0 ml of Solution A and 28.0 ml of Solution B, mix, and dilute 20 times with 0.85% sodium chloride solution. Autoclave and store at room temperature or 4°C.

[0145] 1.4.3 Collect SPF chicken blood containing 1% chicken red blood cells (RBCs), add an equal volume of Alderman's solution, shake well, and store at 4°C for later use. Before use, wash 3 to 5 times with PBS, centrifuging at 2000 r / min for 5 min each time to thoroughly wash away plasma and white blood cells until the supernatant is clear. Prepare a 1% RBC suspension with PBS and store at 4°C for later use.

[0146] 2. Experimental Methods

[0147] 2.1 Experimental Design

[0148] Forty-five SPF chickens were randomly divided into three groups of 15 each. Experimental group 1 received one dose of Newcastle disease live vaccine (LaSota strain) via eye drop and simultaneously received an intramuscular injection of 0.1 ml / bird of rabbit cyst transfer factor as described in Example 3 of this invention. Experimental group 2 received one dose of Newcastle disease live vaccine (La Sota strain) via eye drop and simultaneously received an intramuscular injection of 0.1 ml / bird of existing rabbit cyst transfer factor. An immunization control group was also established (responded only with one dose of Newcastle disease live vaccine (La Sota strain) via eye drop, without injection of rabbit cyst transfer factor), as shown in Table 5.

[0149] Blood samples were collected from each group on days 7, 14, 21 and 28 post-immunization to detect HI antibody (log2). The data were analyzed by one-way ANOVA and the least significant difference test (LSD). P < 0.05 was considered statistically significant.

[0150] Table 5 Experimental Design

[0151]

[0152] 2.2 Newcastle disease vaccine antibody assay in chickens

[0153] 2.2.1 Hemagglutination test

[0154] (1) Add 25 μl of PBS solution to each well of the V-shaped microcoagulation plate.

[0155] (2) Add 25 μl of chicken Newcastle disease virus hemagglutination inhibition test antigen to well 1.

[0156] (3) Serially dilute the Newcastle disease virus hemagglutination inhibition test antigen (NDV) to the reaction plate in 25 μl increments. Do not add NSV hemagglutination inhibition test antigen to well 12; use red blood cells as the control well.

[0157] (4) Add 25 μl of PBS solution to each well.

[0158] (5) Add 25 μl of 1% RBCs to each well.

[0159] (6) Gently tap a small amount of blood coagulation plate to mix the reaction mixture, let it stand at 4°C for 60 min, and determine the result when the RBCs in the control well are significantly button-shaped.

[0160] (7) When determining the hemagglutination test, the reaction plate should be tilted and the RBCs should be observed to see if they flow like teardrops. The maximum dilution factor of the virus that is completely agglutinated (without flowing like teardrops) is the hemagglutination titer of the chicken Newcastle disease virus hemagglutination inhibition test antigen, which is represented by 1 hemagglutination unit (HAU). The hemagglutination titer should then be accurately calculated based on the initial dilution factor.

[0161] 2.2.2 4HAU Newcastle disease virus hemagglutination inhibition test antigen: Based on the titer determined in “2.2.1.1 Hemagglutination test”, calculate the dilution factor of the 4HAU Newcastle disease virus hemagglutination inhibition test antigen.

[0162] 2.2.3 Hemagglutination inhibition test

[0163] (1) Add 25 μl of PBS solution to each well in a V-type microplate.

[0164] (2) Add 25 μl of serum sample to the first well.

[0165] (3) 25 μl of serum was serially diluted horizontally on a blood coagulation plate.

[0166] (4) Add 25 μl of 4HAU antigen to each well and let stand at 4°C for at least 60 min.

[0167] (5) Add 25 μl of 1% RBCs to each well, shake gently to mix, and let stand at 4°C for about 60 min. When the RBCs in the control well show a significant button-like shape, judge the result.

[0168] (6) Each measurement should include positive serum, negative serum and red blood cell controls with known titers.

[0169] 2.2.4 Result Determination

[0170] (1) The test can be established if the error between the positive serum HI antibody (log2) and the known antibody does not exceed 1 titer, the negative serum antibody does not exceed 2log2, and the red blood cell control does not exhibit autoagglutination. Otherwise, the test should be repeated.

[0171] (2) The highest serum dilution factor that completely inhibits the 4HAU chicken Newcastle disease virus hemagglutination inhibition test antigen is used to determine the HI antibody of the serum.

[0172] 3 Results

[0173] The results showed (as shown in Table 6) that on days 14 and 21 post-immunization, the levels of Newcastle disease HI antibodies in experimental groups 1 and 2 were significantly higher than those in the immunized control group (P<0.05). Specifically, the levels of Newcastle disease HI antibodies in experimental group 1 were 2.8 higher than those in the immunized control group, and the levels in experimental group 2 were 1.3 and 1.5 higher than those in the immunized control group, respectively.

[0174] On days 7, 14, 21, and 28 post-immunization, the Newcastle disease HI antibody levels in experimental group 1 were significantly higher than those in experimental group 2 (P<0.05), increasing by 1.0, 1.5, 1.3, and 1.7, respectively. These results indicate that rabbit scleral sac transfer factor can significantly enhance Newcastle disease vaccine-induced antibody levels in chickens, and the immunomodulatory effect of the rabbit scleral sac transfer factor in Example 3 of this invention is significantly higher than that of existing rabbit scleral sac transfer factors.

[0175] Table 6. Experimental Results

[0176]

[0177] Note: Different lowercase superscripts indicate significant differences (P<0.05).

Claims

1. A method for preparing rabbit round capsule transfer factor, characterized in that: It includes the following steps: 1) Select round sacs of healthy rabbits, remove the fascia, muscle and fat tissue on the surface of the rabbit round sacs, wash with water for injection, mince with a meat grinder, mix the water for injection with the minced rabbit round sacs, and homogenize with a colloid mill to obtain a homogenate. 2) After cell disruption, the homogenate was centrifuged to obtain a primary supernatant and a primary precipitate. The primary precipitate was mixed with water for injection adjusted to pH 4.0-5.0 with hydrochloric acid, and homogenized using a colloid mill to obtain a homogenate. 0.01%-0.05% pepsin was added and the mixture was stirred and hydrolyzed at 37°C for 1-5 h. Cell disruption was further performed, and the mixture was centrifuged to obtain a secondary supernatant. The primary and secondary supernatants were combined and then tangentially filtered through a 0.22-0.65 μm filter membrane. The permeate was collected. 3) The permeate is filtered tangentially stepwise using filter membranes with different molecular weight cut-off pore sizes, and the permeate or retentate is collected to obtain the crude rabbit round capsule transfer factor with the required molecular weight range. 4) Mix at least one crude rabbit round capsule transfer factor with a molecular weight range from step 3), wherein the volume ratio of rabbit round capsule transfer factor with a molecular weight of 3 to 5 kDa is 40%-70%, which is the initial product of rabbit round capsule transfer factor. 5) After removing the virus from the initial rabbit round capsule transfer factor product by the virus removal / inactivation method, adjust the pH to 6.5-7.5 and the osmotic pressure to 280-320 mosm / kg, and sterilize it with a 0.1-0.22 μm sterilization filter to obtain rabbit round capsule transfer factor.

2. The method for preparing rabbit round capsule transfer factor according to claim 1, characterized in that: Step 3) is as follows: 3-1) Preparation of crude rabbit round capsule transfer factor less than 10 kDa: The permeate from step 2) was tangentially filtered using a 10 kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 10 kDa. 3-2) Preparation of crude rabbit round capsule transfer factor less than 8 kDa: The permeate from step 2) was tangentially filtered using an 8 kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 8 kDa. 3-3) Preparation of crude rabbit round capsule transfer factor less than 5 kDa: The permeate from step 2) was tangentially filtered using a 5 kDa filter membrane, and the permeate was collected, which is the crude rabbit round capsule transfer factor less than 5 kDa. 3-4) Preparation of crude rabbit round capsule transfer factor of 8 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3-1) is filtered tangentially using an 8 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 8 to 10 kDa. 3-5) Preparation of crude rabbit round capsule transfer factor of 5 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3-1) is filtered tangentially using a 5 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 5 to 10 kDa. 3-6) Preparation of crude rabbit round capsule transfer factor of 3 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3-1) is filtered tangentially using a 3 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 10 kDa. 3-7) Preparation of crude rabbit round capsule transfer factor of 1 to 10 kDa: The crude rabbit round capsule transfer factor of less than 10 kDa in step 3-1) is filtered tangentially using a 1 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 10 kDa. 3-8) Preparation of crude rabbit round capsule transfer factor of 5 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 3-2) is filtered tangentially using a 5 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 5 to 8 kDa. Alternatively, the crude 5 to 10 kDa rabbit cyst transfer factor from steps 3-5) can be tangentially filtered using an 8 KD filter membrane, and the permeate can be collected to obtain the crude 5 to 8 kDa rabbit cyst transfer factor. 3-9) Preparation of crude rabbit round capsule transfer factor of 3 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 3-2) is filtered tangentially using a 3 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 8 kDa. Alternatively, the crude 3 to 10 kDa rabbit cyst transfer factor from steps 3-6) can be tangentially filtered using an 8 KD filter membrane, and the permeate can be collected to obtain the crude 3 to 8 kDa rabbit cyst transfer factor. 3-10) Preparation of crude rabbit round capsule transfer factor of 1 to 8 kDa: The crude rabbit round capsule transfer factor of less than 8 kDa in step 3-2) is filtered tangentially using a 1 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 8 kDa. Alternatively, the crude 1 to 10 kDa rabbit cyst transfer factor from steps 3-7) can be tangentially filtered using an 8 KD filter membrane, and the permeate can be collected to obtain the crude 1 to 8 kDa rabbit cyst transfer factor. 3-11) Preparation of crude rabbit round capsule transfer factor of 3 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 3-3) is filtered tangentially using a 3 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 3 to 5 kDa. Alternatively, the crude rabbit cyst transfer factor of 3 to 10 kDa in step 3-6) or the crude rabbit cyst transfer factor of 3 to 8 kDa in step 3-9) can be tangentially filtered using a 5 KD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 3 to 5 kDa. 3-12) Preparation of crude rabbit round capsule transfer factor of 1 to 5 kDa: The crude rabbit round capsule transfer factor of less than 5 kDa in step 3-3) is filtered tangentially using a 1 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 5 kDa. Alternatively, the crude rabbit cyst transfer factor of 1 to 10 kDa from step 3-7) or the crude rabbit cyst transfer factor of 1 to 8 kDa from step 3-10) can be tangentially filtered using a 5 KD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 1 to 5 kDa. 3-13) Preparation of crude rabbit cyst transfer factor less than 3 kDa: The crude rabbit cyst transfer factor less than 10 kDa in step 3-1), the crude rabbit cyst transfer factor less than 8 kDa in step 3-2), or the crude rabbit cyst transfer factor less than 5 kDa in step 3-5) is subjected to tangential flow filtration using a 3 KD filter membrane. The permeate is collected, which is the crude rabbit cyst transfer factor less than 3 kDa. Alternatively, the permeate from step 2) can be tangentially filtered using a 30-500 kDa filter membrane, and the permeate can be collected and then tangentially filtered again using a 3KD filter membrane. The collected permeate is the crude rabbit round capsule transfer factor with a value of less than 3 kDa. 3-14) Preparation of crude rabbit round capsule transfer factor of 1 to 3 kDa: The crude rabbit round capsule transfer factor of less than 3 kDa in step 3-13) is filtered tangentially using a 1 KD filter membrane, and the retentate is collected, which is the crude rabbit round capsule transfer factor of 1 to 3 kDa. Alternatively, the crude rabbit cyst transfer factor of 1 to 10 kDa from step 3-7), the crude rabbit cyst transfer factor of 1 to 8 kDa from step 3-10), or the crude rabbit cyst transfer factor of 1 to 5 kDa from step 3-12) can be tangentially filtered using a 3 KD filter membrane, and the permeate can be collected to obtain the crude rabbit cyst transfer factor of 1 to 3 kDa. 3-15) Preparation of crude rabbit cyst transfer factor less than 1 kDa: The crude rabbit cyst transfer factor less than 10 kDa in step 3-1), the crude rabbit cyst transfer factor less than 8 kDa in step 3-2), the crude rabbit cyst transfer factor less than 5 kDa in step 3-3), or the crude rabbit cyst transfer factor less than 3 kDa in step 3-13) are subjected to tangential flow filtration using a 1 KD filter membrane. The permeate is collected, which is the crude rabbit cyst transfer factor less than 1 kDa. Alternatively, the permeate from step 2) can be tangentially filtered using a 30-300 kDa filter membrane, and the permeate can be collected and then tangentially filtered again using a 1 kD filter membrane. The collected permeate is the crude rabbit round capsule transfer factor with a value of less than 1 kDa.

3. The method for preparing rabbit round capsule transfer factor according to claim 1, characterized in that: In step 1), if a healthy rabbit is positive for antibodies after being immunized with a vaccine or stimulated by an antigen, then in step 5), rabbit round vesicle transfer factor specific to that vaccine or antigen is obtained; conversely, if a healthy rabbit is negative for antibodies without being immunized with a vaccine or stimulated by an antigen, then in step 5), non-specific rabbit round vesicle transfer factor is obtained.

4. The method for preparing rabbit round capsule transfer factor according to claim 1, characterized in that: In step 2), the cell disruption method includes at least one of the following methods: ① repeated freeze-thaw disruption; ② disruption using a high-pressure homogenizer; ③ disruption using an ultrasonic cell disruptor.

5. The method for preparing rabbit round capsule transfer factor according to claim 1, characterized in that: In step 5), the virus removal / inactivation methods include at least two of the following methods: ① Low pH incubation method: react at 4-25℃ for 2 h-1 d at pH 2.0-4.0; ② Membrane filtration method: using a 15-45 nm virus removal filter; ③ β-propiolactone inactivation method: Inactivation at 0.001%-0.025% concentration at 4℃ for 6-24 h, followed by hydrolysis at 37℃ for 2-8 h; ④ Formaldehyde inactivation method: Inactivate at 37℃ for 6-12 hours at a concentration of 0.01%-0.05%.

6. The application of the rabbit round capsule transfer factor obtained by the preparation method according to claim 1 in the preparation of drugs that improve the level of animal specific immunity.

Images