CN114805541A

CN114805541A - Preparation method of pig thymosin

Info

Publication number: CN114805541A
Application number: CN202210594601.4A
Authority: CN
Inventors: 徐磊; 钟敏; 万培伟; 王均辉; 王舒宁; 杨慧; 刘毅发; 谢杼倢; 廖惠珍; 钟云钦; 王宗冬; 陈雷; 朱海侠; 黄瑜; 张渊魁
Original assignee: Fujian Fuyu Agricultural Technology Development Co ltd; Fujian Xingbang Biotechnology Co ltd; Fuzhou Zhongwei Biotechnology Co ltd; Present Fuzhou Bio Tech Co ltd; Fujian Vocational College of Agriculture
Current assignee: Fujian Fuyu Agricultural Technology Development Co ltd; Fujian Xingbang Biotechnology Co ltd; Fuzhou Zhongwei Biotechnology Co ltd; Present Fuzhou Bio Tech Co ltd; Fujian Vocational College of Agriculture
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-07-29

Abstract

The invention discloses a preparation method of pig thymosin, which comprises the following steps of 1) mixing water for injection or water for injection with the pH value adjusted to 1.5-6.5 by hydrochloric acid with minced pig thymus, and homogenizing by a colloid mill to obtain homogenate; 2) breaking the homogenate by cells, insulating in water, centrifuging and collecting the centrifugal supernatant; 3) performing tangential flow filtration on the harvested centrifugal supernatant by using a filter membrane, and collecting a permeate; 4) gradually performing tangential flow filtration on the permeate by using filter membranes with different cut-off pore diameters and different molecular weights, and collecting the permeate to obtain a crude product of the porcine thymosin with the required molecular weight range; 5) mixing at least one thymosin crude product with a molecular weight range in the step 4) to obtain a thymosin crude product; 6) and (3) after virus is removed from the primary product of the porcine thymosin, regulating the pH and osmotic pressure, and filtering and sterilizing to obtain the porcine thymosin. The invention adopts step-by-step tangential flow filtration, establishes an accurate and effective pig thymosin separation and concentration technology and a small molecule purification technology, and selects filter membranes with different cut-off pore diameters and different molecular weights to prepare the pig thymosin in the required molecular weight range according to different process technical flows.

Description

Preparation method of pig thymosin

Technical Field

The invention belongs to the field of biological products, and particularly relates to a preparation method of pig thymosin.

Background

The thymus is an important immune organ of animals and can secrete a plurality of important hormones, such as: thymosins (Thymosin), Thymic humoral factors (Thymus humoraral factor), thymopoietins (thymopoietin), and thymokines (Thymic factor). The thymosin is a group of polypeptides (composition) secreted by thymic tissue epithelial cells and having biological activity, and plays an important role in the adjuvant therapy of various primary or secondary T cell deficiency diseases, some autoimmune diseases and various cell immunologic hypofunction diseases.

Thymus was found to play an important role in lymphocyte differentiation, maturation and immunological activity in mice and rabbits in 1961 by Miller and Archer, respectively, and thereafter many scholars have successively isolated a mixture of thymosin peptides from the thymus or serum of human embryos, calf, sheep, pig, etc. In 1972, Golden separated thymosin fraction 5(TF5) from calf thymus and obtained alpha by isoelectric focusing electrophoresis _1-10 、β _1-5 And gamma, 3 regions, 15 polypeptides. Currently, a single thymosin peptide is prepared by genetic engineering, such as: alpha is alpha ₁ Thymopentin or derivatives thereof, and the like. Although the use of genetic engineering to produce a single thymosin peptide has been used clinically, the thymosin peptide mixture extracted from thymus still has its advantages because of the different thymosin peptidesThe effect on different immune cells and different development stages thereof is different, and various peptide components can play a synergistic role.

At present, the preparation method of the porcine thymosin has the following defects:

(1) thymosin is a group of polypeptides (compositions) secreted by epithelial cells of the thymus tissue with biological activity, and the effect of different thymosin components on different immune cells and different stages of development thereof is different. Therefore, the components of the thymosin have different molecular weights, and have different biological activities, preparation qualities, application doses and clinical applications. The prior thymic peptide manufacturing technology lacks an effective thymic peptide separation and concentration technology and a small molecule purification technology, and does not have a preparation method of thymic peptides with different molecular weights, so that the thymic peptides with different molecular weight compositions can not be obtained. These limit the study of the biological activity, the study of the mechanism of action and the effective clinical application of thymosin.

(2) The thymosin is derived from animal thymus, so animal virus can be transmitted by thymosin. Wherein, bovine viral diarrhea virus, porcine parvovirus, classical swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus are important epidemic diseases which seriously endanger the swine industry in China. To ensure the safety of the porcine thymosin, the porcine thymus must be selected and tested. The prior art for manufacturing the pig thymosin lacks of carrying out strict and systematic exogenous virus inspection on the pig thymus, particularly lacks of inspection on African swine fever virus of new pig disease in China, so that the prepared pig thymosin has larger virus safety risk.

(3) Due to the technical level limitation, there may exist swine-origin viruses whose characteristics cannot be found or understood. In the preparation of the porcine thymosin, the potential unknown virus in the porcine thymus still exposes the porcine thymosin to virus safety risks. Therefore, the virus removal/inactivation process is an important and necessary means for ensuring the safety of the porcine thymosin. The traditional pig thymosin manufacturing technology mainly adopts a 5-10kDa ultrafiltration membrane for ultrafiltration or dialysis bag for dialysis to directly obtain a finished product of the pig thymosin, the technological process is not treated by a virus removal/inactivation method, the ultrafiltration membrane ultrafiltration and the dialysis bag for dialysis both have a certain virus interception and removal effect, but are limited to the structural characteristics and the filtration technological properties of the ultrafiltration membrane and the dialysis bag, and neither the ultrafiltration membrane ultrafiltration nor the dialysis bag for ultrafiltration is a safe, reliable and easily verified virus removal/inactivation method, so that the prepared pig thymosin has a greater virus safety risk.

(4) In order to ensure the safety of the pig thymosin, measures such as inspection and selection of the pig thymus and virus removal/inactivation treatment of the pig thymosin are taken, and strict and systematic exogenous virus inspection of the prepared pig thymosin is also an important link for ensuring the safety of the pig thymosin. The prior art for manufacturing the pig thymosin lacks of carrying out strict and systematic exogenous virus inspection on the pig thymus, particularly lacks of inspection on African swine fever virus of new pig disease in China, so that the prepared pig thymosin has larger virus safety risk.

(5) The conventional thymosin manufacturing technology is characterized in that homogenate is treated by keeping the temperature of 70-85 ℃ for 5-30min so as to remove impure proteins to realize clarification, and then 5-10kDa ultrafiltration membrane ultrafiltration is carried out to obtain the thymosin. The prior art for preparing the thymosin has the defect that the polypeptide structure and the activity of the thymosin are greatly damaged by heat treatment at the temperature of 70-85 ℃. The reason why the homogenate needs to be subjected to heat treatment at 70-85 ℃ before centrifugation is that if the homogenate is subjected to ultrafiltration directly after centrifugation, the filtration efficiency is extremely poor, so that the ultrafiltration membrane can be almost used only once.

(6) Although thymus is a waste product in the processing process, thymus still does not belong to an unlimited supply of raw material resources. The prior thymus peptide manufacturing technology has poor use efficiency on thymus, only selects centrifugal supernatant after homogenate centrifugation to prepare the thymus peptide, and abandons centrifugal precipitates. In addition, the existing thymus peptide manufacturing technology lacks an osmotic pressure adjusting process, and isotonic thymus peptide cannot be obtained, so that the application effect and the application range of the thymus peptide are limited.

Disclosure of Invention

The invention aims to overcome the defects of the background technology and provide a preparation method of pig thymosin without virus safety risk, and the pig thymosin with specific molecular weight can be obtained by the method.

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

a preparation method of pig thymosin comprises the following steps:

1) selecting thymus gland of healthy pig, removing fascia, muscle and fat tissue on the surface of the thymus gland, cleaning with water for injection, mincing with mincer, mixing water for injection or water for injection with pH value adjusted to 1.5-6.5 with hydrochloric acid with minced thymus gland, and homogenizing with colloid mill to obtain homogenate;

2) breaking the homogenate, keeping the temperature for 0.5-2h in water at 60 ℃, and centrifuging to respectively obtain primary centrifugal supernatant and centrifugal precipitate;

or further mixing the centrifuged precipitate with water for injection or water for injection with pH value adjusted to 1.5-6.5 by hydrochloric acid, homogenizing by colloid mill to obtain homogenate, crushing cells, keeping at 60 deg.C for 0.5-2 hr, centrifuging to obtain secondary centrifuged supernatant, and mixing the primary and secondary centrifuged supernatants;

3) performing tangential flow filtration on the harvested centrifugal supernatant through a 0.1-0.45 mu m filter membrane, and collecting permeate;

4) tangential flow filtration is carried out on part of the permeate in the step 3) by adopting a 5-10kDa filter membrane, and the permeate is collected, namely a crude product of the porcine thymosin with the molecular weight of less than 5-10 kDa;

performing tangential flow filtration on part of the permeate in the step 3) by using a 10-1000kDa filter membrane, collecting the permeate, performing tangential flow filtration by using a 1-3kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight of less than 1-3 kDa;

5) mixing at least one of the crude products of the porcine thymosin in the molecular weight range in the step 4) to obtain a primary product of the porcine thymosin;

6) after virus removal is carried out on the primary product of the porcine thymosin by a virus removal/inactivation method, the pH is adjusted to 6.5-7.5, the osmotic pressure is adjusted to 280-320mosm/kg, and a sterilization filter with the diameter of 0.1-0.22 mu m is adopted for sterilization, thus obtaining the porcine thymosin.

Furthermore, in the step 1), the pig thymus should not be polluted by bovine viral diarrhea virus, porcine parvovirus, classical swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus, and the exogenous virus inspection method is detailed in the attached notes I.

In step 2), the cell disruption method comprises at least one of the following methods: freezing and thawing repeatedly and crushing; crushing by a high-pressure homogenizer; and crushing by using an ultrasonic cell crusher.

In step 6), the virus removal/inactivation method includes at least two of the following methods: firstly, a low pH incubation method (the reaction is carried out for 2h-1d at 4-25 ℃ when the pH value is 2.0-4.0); membrane filtration (15-45nm virus-removing filter filtration); ③ a beta-propiolactone inactivation method (0.001 to 0.025 percent of the concentration, inactivation is carried out at 4 ℃ for 6 to 24 hours, and then hydrolysis is carried out at 37 ℃ for 2 to 8 hours); fourthly, formaldehyde inactivation (inactivation is carried out for 6 to 12 hours at 37 ℃ when the concentration is 0.01 to 0.05 percent); pasteurization (inactivation at 60 deg.C for 3-5 h).

In the step 6), the porcine thymosin should not be polluted by bovine viral diarrhea virus, hog cholera virus, porcine parvovirus, porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus and African swine fever virus, and the exogenous virus inspection method is detailed in the attached notes I.

In the prepared finished product of the porcine thymosin, when the volume ratio of the porcine thymosin with the molecular weight less than 1kDa is 30-70%, the porcine thymosin is suitable for improving the activity of animal lymphocytes.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts step-by-step tangential flow filtration, establishes an accurate and effective pig thymosin separation and concentration technology and a small molecule purification technology, selects filter membranes with different cut-off pore diameters and different molecular weights, can prepare pig thymosin crude products with different molecular weights less than 1-10 kDa according to different process technical flows, mixes at least one pig thymosin crude product with a molecular weight range according to different proportions, and removes bacteria after virus removal/inactivation treatment, PH and osmotic pressure regulation, thus obtaining the pig thymosin.

Thymosin peptides of different molecular weights have different biological activities. Compared with the pig thymosin prepared by the existing pig thymosin preparation technology, the pig thymosin prepared by the invention has different proportions of different molecular weight components, different biological activities and different dosage and clinical effects, and provides a technical basis for the research of the biological activity, the research of the action mechanism and the effective clinical application of the thymosin.

2. The quality of the pig thymus is the primary restriction factor of the pig thymus peptide, the quality and safety of the pig thymus peptide are seriously affected by the infection of the exogenous virus, the most important quality monitoring before the formal preparation of the pig thymus peptide is carried out when the pig thymus is infected by the exogenous virus, the quality monitoring of the pig thymus is the precondition for producing the qualified pig thymus peptide, and the method plays an important role in producing safe and effective products. The prior art for manufacturing the porcine thymosin lacks of carrying out strict and systematic exogenous virus inspection on the porcine thymus. Compared with the prior art for manufacturing the pig thymosin, the invention establishes a strict systematic method for detecting the raw material pig thymosin by surrounding bovine viral diarrhea virus, porcine parvovirus, classical swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, pseudorabies virus and African swine fever virus, particularly adds the raw material pig thymosin exogenous virus detection to the new-born African swine fever virus in China, strictly detects and screens the pig thymosin, ensures the safety of the pig thymosin and strictly controls the virus safety risk of the prepared pig thymosin.

3. The pig thymus is subjected to the processes of exogenous virus inspection screening, tangential flow filtration and the like to prepare a pig thymus peptide crude product, and the pig thymus peptide crude product still can be polluted by exogenous viruses which are mistakenly introduced or potential unknown viruses. In order to eliminate the possible foreign virus remained in the crude product of the porcine thymosin and prevent the virus from entering the finished product of the porcine thymosin, the crude product of the porcine thymosin is treated by a virus removal/inactivation method. The existing pig thymosin manufacturing technology mostly adopts ultrafiltration membrane ultrafiltration or dialysis bag dialysis interception which is insufficient in safety and reliability and difficult to verify to remove viruses, so that the possibility that some viruses cannot be completely removed exists, the safety of the pig thymosin cannot be absolutely guaranteed, and the prepared pig thymosin has a large virus safety risk. The virus removing/inactivating method for the porcine thymosin, which is disclosed by the invention, is used for carrying out verification and evaluation research on the virus removing/inactivating method for the porcine thymosin, and screening and establishing the virus removing/inactivating method which has the advantages of wide virus inactivating range, good effect and qualified verification and can keep the efficacy and stability of the porcine thymosin from a pasteurization method, a dry heat method, an organic solvent/detergent (S/D) treatment method, a membrane filtration method, a low pH incubation method, a beta-propiolactone inactivation method, a formaldehyde inactivation method, a diethylene imine inactivation method, an octanoic acid treatment method, a photochemical method, a depth filtration technology, a chromatographic technology and the like. Compared with the prior art for manufacturing the porcine thymosin, the invention comprises at least two virus removing/inactivating methods as follows: firstly, a low pH incubation method (reaction at 4-25 ℃ for 2h-1d when the pH is 2.0-4.0); membrane filtration (15-45nm virus-removing filter filtration); ③ a beta-propiolactone inactivation method (0.001 to 0.025 percent of the concentration, inactivation is carried out at 4 ℃ for 6 to 24 hours, and then hydrolysis is carried out at 37 ℃ for 2 to 8 hours); fourthly, formaldehyde inactivation (inactivation is carried out for 6 to 12 hours at 37 ℃ when the concentration is 0.01 to 0.05 percent); pasteurization (inactivation at 60 deg.C for 3-5 h). It is clear that two or more than two virus removal/inactivation methods with different mechanisms are required to be respectively adopted in the production process of the pig thymosin so as to ensure the complete inactivation/removal of the virus and the safety of the pig thymosin.

4. Exogenous viral contamination will seriously affect the quality and safety of thymosin, and exogenous virus is not allowed to exist in any animal-derived thymosin. Therefore, the strict and systematic exogenous virus inspection of the prepared porcine thymosin is an important link for ensuring the safety of the porcine thymosin. The prior thymus peptide manufacturing technology lacks of carrying out strict and systematic exogenous virus inspection on the thymus of the pig, particularly lacks of inspecting the African swine fever virus of new pig disease in China, so that the prepared thymus of the pig has larger virus safety risk. According to the invention, exogenous virus detection is firstly carried out on the raw material pig thymus, so that the raw material pig thymus is ensured to be free from exogenous virus pollution. And a 1-10KD filter membrane is used for tangential flow ultrafiltration or nanofiltration, so that the safety of the crude product of the porcine thymosin is further ensured. Subsequently, the virus is inactivated using at least two virus removal/inactivation methods to inactivate the virus if present. Compared with the prior art for manufacturing the pig thymosin, the invention establishes a strict system method for inspecting the pig thymosin exogenous virus around bovine viral diarrhea virus, porcine parvovirus, swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus, particularly newly adds the exogenous virus inspection of the pig thymosin to new swine fever African swine fever virus in China, and really ensures that the pig thymosin does not have exogenous virus pollution.

5. Thymosin, as a polypeptide composition, is more sensitive to temperature. In the existing thymus peptide manufacturing technology, the homogenate is kept at 70-85 ℃ for 5-30min to be subjected to heat treatment, so that the clarification effect of removing foreign proteins is realized, the homogenate is beneficial to being directly filtered and separated from the thymus peptide after being centrifuged, but the polypeptide structure and the activity of the thymus peptide are greatly damaged. According to the invention, the heat treatment temperature is reduced, and after the homogenate is subjected to water insulation and heat preservation at 60 ℃ for 0.5-2h, the structural integrity and activity maintenance of the thymosin are not influenced, and the subsequent filtering and separation of the thymosin are facilitated. In addition, 60 ℃ meets the temperature requirements of pasteurization, thereby removing/inactivating viruses.

6. Although thymus is a waste product in the processing process, thymus still does not belong to an unlimited supply of raw material resources. The existing pig thymus peptide manufacturing technology has poor use efficiency on pig thymus, and centrifugal supernatant is selected for preparing the pig thymus peptide only after homogenate centrifugation, while centrifugal precipitate is abandoned. In the invention, the centrifugal precipitate is subjected to secondary extraction: further mixing the centrifugal precipitate with water for injection or water for injection with pH value adjusted to 1.5-6.5 by hydrochloric acid, homogenizing by colloid mill to obtain homogenate, breaking cells, keeping at 60 deg.C for 0.5-2 hr, and centrifuging to obtain centrifugal supernatant for preparing pig thymosin. The invention adopts the water for injection with the pH value of 1.5-6.5 to mix and homogenize, and the temperature is kept for 0.5-2 hours in a water-proof way at 60 ℃, thereby being beneficial to removing/inactivating viruses and inhibiting enzyme activity, better retaining active substances in homogenate, being beneficial to high-efficiency extraction of the porcine thymosin, realizing secondary extraction of centrifugal precipitate and improving the yield of the porcine thymosin.

7. The existing thymus peptide manufacturing technology lacks an osmotic pressure regulation process, cannot obtain isotonic thymus peptide, cannot combine immunization, combined freeze-drying and co-injection with attenuated vaccine, and limits the application effect and the application range of the thymus peptide. In the invention, the osmotic pressure regulation technology is realized, the osmotic pressure of the primary product of the porcine thymosin is regulated to be an isotonic solution (280-320mosm/kg), the combined immunization, the combined freeze-drying and the co-injection of the porcine thymosin and the attenuated vaccine are realized, the application effect of the porcine thymosin is improved, and the application range of the porcine thymosin is expanded.

Drawings

FIG. 1 shows the results of the activity measurement of the de-E receptor method in comparative example 1.

FIG. 2 shows the test results for classical swine fever virus of comparative example 2.

Detailed Description

Example 1

A preparation method of pig thymosin comprises the following steps:

1) selecting thymus of healthy pigs, wherein the thymus of the healthy pigs is free from pollution of bovine viral diarrhea virus, porcine parvovirus, hog cholera virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus, removing fascia, muscle and fat tissues on the surface of the thymus of the healthy pigs, cleaning the thymus of the healthy pigs with water for injection, mincing the thymus of the healthy pigs by a meat mincer, mixing the water for injection with the minced thymus of the healthy pigs, and homogenizing the mixture by a colloid mill to obtain homogenate;

2) the homogenate is subjected to cell disruption, water insulation and heat preservation at 60 ℃ for 0.5h, and then centrifugation is carried out to obtain centrifugal supernatant and centrifugal precipitate respectively. Further mixing the centrifugal precipitate with water for injection, homogenizing by colloid mill to obtain homogenate, breaking cells, keeping at 60 deg.C in water for 1h, centrifuging to obtain centrifugal supernatant, and mixing the supernatants for 2 times.

In the step, the cell disruption method comprises the following steps: the first cell disruption is implemented by using an ultrasonic cell disruption instrument, and the second cell disruption is implemented by using repeated freeze thawing.

3) The harvested centrifugation supernatant was subjected to tangential flow filtration through a 0.22 μm filter and the permeate was collected.

4) Tangential flow filtration is carried out on part of permeate in the step 3) by adopting a 5kDa filter membrane, and the permeate is collected, namely the porcine thymosin crude product with the molecular weight less than 5 kDa; and performing tangential flow filtration on part of the permeate in the step 3) by using a 500kDa filter membrane, collecting the permeate, performing tangential flow filtration by using a 1kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight less than 1 kDa.

5) Mixing the pig thymosin crude product with the molecular weight of less than 5kDa and the pig thymosin crude product with the molecular weight of less than 1kDa in the step 4), wherein the mixing ratio is as follows: the crude product of the porcine thymosin with the molecular weight less than 5kDa accounts for 70 percent of the volume of the mixed solution, and the crude product of the porcine thymosin with the molecular weight less than 1kDa accounts for 30 percent of the volume of the mixed solution, namely the crude product of the porcine thymosin.

6) And (3) removing viruses of the primary product of the porcine thymosin by a virus removal/inactivation method, adjusting the pH to 6.6, adjusting the osmotic pressure to 281mosm/kg, and sterilizing by a 0.1-micrometer sterilizing filter to obtain the porcine thymosin.

The virus removing/inactivating method comprises the following two methods: beta-propiolactone inactivation (0.001% concentration, 4 ℃ inactivation for 24h, 37 ℃ hydrolysis for 2 h); pasteurization (inactivation at 60 ℃ for 3 h).

The obtained porcine thymosin has no pollution of bovine viral diarrhea virus, swine fever virus, porcine parvovirus, porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus and African swine fever virus.

The product of the embodiment is especially suitable for improving the activity of animal lymphocyte (the volume ratio of the pig thymosin with the molecular weight less than 1kDa is 30-70%).

Example 2

A preparation method of pig thymosin comprises the following steps:

1) selecting thymus of healthy pigs, wherein the thymus of the healthy pigs is free from pollution of bovine viral diarrhea virus, porcine parvovirus, hog cholera virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus, removing fascia, muscle and fat tissues on the surface of the thymus of the healthy pigs, cleaning the thymus of the healthy pigs with water for injection, mincing the thymus of the healthy pigs by a mincer, mixing the water for injection with hydrochloric acid of which the pH value is adjusted to 1.5 with the minced thymus of the healthy pigs, and homogenizing the mixture by a colloid mill to obtain homogenate.

2) And (3) breaking the homogenate by cells (repeated freeze-thaw disruption method), insulating water at 60 ℃ for 2 hours, and centrifuging to obtain a centrifugal supernatant and a centrifugal precipitate respectively. Further mixing the centrifuged precipitate with water for injection adjusted to pH 6.5 with hydrochloric acid, homogenizing by colloid mill to obtain homogenate, performing cell disruption (repeated freeze-thaw disruption method), keeping at 60 deg.C for 2 hr, and centrifuging to obtain centrifuged supernatant. The centrifugation supernatants were combined.

3) The harvested centrifugation supernatant was subjected to tangential flow filtration through a 0.45 μm filter and the permeate was collected.

4) And 3) performing tangential flow filtration on part of the permeate in the step 3) by adopting a 10kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight less than 10 kDa. And performing tangential flow filtration on part of the permeate in the step 3) by using a 1000kDa filter membrane, collecting the permeate, performing tangential flow filtration by using a 1kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight less than 1 kDa.

5) Mixing the pig thymosin crude product with the molecular weight of less than 10kDa and the pig thymosin crude product with the molecular weight of less than 1kDa in the step 4), wherein the mixing ratio is as follows: the crude product of the porcine thymosin with the molecular weight less than 10kDa accounts for 30 percent of the volume of the mixed solution, and the crude product of the porcine thymosin with the molecular weight less than 1kDa accounts for 70 percent of the volume of the mixed solution, namely the crude product of the porcine thymosin.

6) And (3) removing viruses from the primary product of the porcine thymosin by a virus removal/inactivation method, adjusting the pH to 7.3, adjusting the osmotic pressure to 310mosm/kg, and sterilizing by a 0.22 mu m sterilizing filter to obtain the porcine thymosin.

The virus removing/inactivating method comprises the following two methods: low pH incubation (reaction at 4 ℃ for 1d at pH 2.0); pasteurization (5 h inactivation at 60 ℃).

Example 3

A preparation method of pig thymosin comprises the following steps:

1) selecting thymus of healthy pigs, wherein the thymus of the healthy pigs is free from pollution of bovine viral diarrhea virus, porcine parvovirus, hog cholera virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus and African swine fever virus, removing fascia, muscle and fat tissues on the surface of the thymus of the healthy pigs, cleaning the thymus of the healthy pigs with water for injection, mincing the thymus of the healthy pigs by a meat mincer, mixing the water for injection with the minced thymus of the healthy pigs, and homogenizing the mixture by a colloid mill to obtain homogenate.

2) The homogenate is subjected to cell disruption, water insulation and heat preservation at 60 ℃ for 1h, and then centrifugation is carried out to obtain centrifugal supernatant and centrifugal precipitate respectively. Further mixing the centrifuged precipitate with water for injection adjusted to pH2.0 with hydrochloric acid, homogenizing by colloid mill to obtain homogenate, breaking cells, keeping at 60 deg.C for 0.5 hr, and centrifuging to obtain centrifuged supernatant. The centrifugation supernatants were combined.

Wherein, the cell disruption method comprises the following steps: the first cell disruption adopts repeated freeze thawing disruption, and the second cell disruption adopts high pressure homogenizer disruption.

3) The harvested centrifugation supernatant was subjected to tangential flow filtration through a 0.1 μm filter and the permeate was collected.

4) Performing tangential flow filtration on the permeate in the step 3) by using an 8kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight less than 8 kDa. Or, performing tangential flow filtration on the permeate in the step 3) by using a 300kDa filter membrane, collecting the permeate, performing tangential flow filtration by using a 1kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight of less than 1 kDa.

5) Mixing the pig thymosin crude product with the molecular weight of less than 8kDa in the step 4) with the pig thymosin crude product with the molecular weight of less than 1kDa, wherein the mixing ratio is as follows: the crude product of the porcine thymosin with the molecular weight less than 8kDa accounts for 30 percent of the volume of the mixed solution, and the crude product of the porcine thymosin with the molecular weight less than 1kDa accounts for 70 percent of the volume of the mixed solution, namely the crude product of the porcine thymosin.

6) And (3) removing viruses of the primary product of the porcine thymosin by a virus removal/inactivation method, adjusting the pH to 6.9, adjusting the osmotic pressure to 292mosm/kg, and sterilizing by a 0.1-micrometer sterilizing filter to obtain the porcine thymosin.

The virus removing/inactivating method comprises the following two methods: ③ inactivating the formaldehyde (0.01 percent of the formaldehyde is inactivated for 6 hours at 37 ℃; ② a membrane filtration method (45nm virus-removing filter filtration).

The following comparative experiments were conducted on the porcine thymosin peptide prepared by the present invention and the existing porcine thymosin peptide by comparative examples 1-3. The conventional porcine thymosin used in the following experiments was prepared according to the Chinese invention patent with the patent application number of 201510158633.X and the patent name of 'preparation and extraction method of thymosin and product of safety of medication'.

Comparative example 1

Comparative experiment for determining the Activity of the porcine thymosin prepared in example 1 of the invention and the Activity of the conventional porcine thymosin by the De-E receptor method

The method is based on that the thymosin can make the thymus T cell after removing E receptor restore its E receptor function so as to reflect the biological activity of thymosin.

1. Reagent

(1) Hank's solution is prepared by mixing 0.3% potassium dihydrogen phosphate solution, 0.76% disodium hydrogen phosphate solution, 2% potassium chloride solution and 20% sodium chloride solution in sequence according to the proportion of 20:20:20:40, adding 1g of glucose, dissolving and mixing uniformly, diluting with water to 1000ml, and adjusting the pH value to 7.2-7.3 with 4% sodium bicarbonate solution (prepared at the time of use).

(2) Dissolving 0.420g sodium chloride, 0.055g citric acid, 0.766g sodium citrate and 2.05g glucose in water, diluting to 100ml, and sterilizing.

(3) The separation liquid is lymphocyte separation liquid.

(4) 5ml of sheep venous blood is taken from sheep blood, added into 5ml of the donkey-hide gelatin solution and stored in a refrigerator.

(5) The fixing solution is prepared by mixing 25% glutaraldehyde solution, 3.5% sodium bicarbonate solution and Hank's solution in a ratio of 1:1:38 in sequence.

(6) Taking 0.5g of giemsa dye in the stock solution of the giemsa staining solution, adding 33ml of glycerol, heating at 55-60 ℃ until the giemsa dye is dissolved, cooling to room temperature, adding 33ml of methanol, standing at room temperature for 24 hours, and filtering with filter paper to obtain a filtrate, namely the stock solution. Sealing and storing at room temperature. (7) Taking 2ml of stock solution of the giemsa staining solution, adding 6ml of Hank's solution, shaking uniformly, centrifuging at 1500 rpm for 10 minutes, and taking supernatant for later use.

2. Method of operation

(1) Preparation of E-receptor-free thymus T cell suspension fresh pig thymus is taken, fat is removed, shearing is carried out, an appropriate amount of Hank's solution is added to form cell suspension, filtering is carried out through a 100-mesh sieve, centrifugation is carried out for 3-5 minutes at 1500 revolutions per minute, supernatant is discarded, a small amount of Hank's solution is added to the solution and stirred evenly, the solution is added into a centrifugal tube with 1/3 filter solution separating medium, centrifugation is carried out for 20 minutes at 2000 revolutions per minute, thymocytes in the middle layer are carefully sucked out, the solution is placed into another centrifugal tube, an appropriate amount of Hank's solution is added to the centrifugal tube, washing is carried out, shaking is carried out evenly, centrifugation is carried out for 3-5 minutes at 1500 revolutions per minute, supernatant is discarded, after washing is carried out once, an appropriate amount of Hank's solution is added into sediment, even mixing is carried out, and heat preservation is carried out in a constant-temperature water bath at 45 ℃ for 30 minutes (shaking is carried out once every 5 minutes). Centrifuging for 3-5 minutes at 1500 revolutions per minute, discarding the supernatant, adding a proper amount of Hank's solution, uniformly mixing, placing in a 45-DEG C constant-temperature water bath, preserving the heat for 30 minutes, taking out, centrifuging for 3-5 minutes at 1500 revolutions per minute, and discarding the supernatant. Washed three times with Hank's solution (same procedure as above), diluted appropriately with Hank's solution and counted to a final concentration of 1ml each (3X 10) ⁶ )～(5×10 ⁶ ) And (4) cells.

(2) Preparation of sheep erythrocyte suspension an appropriate amount of sheep blood was taken and washed three times with an appropriate amount of Hank's solution (supra). And (4) discarding the supernatant, adding a proper amount of Hank's solution to dilute and count so that the final concentration is 8-10 times of the concentration of the E-removed receptor thymic T cell suspension.

(3) Preparation of test solution the porcine thymosin prepared in example 1 of the present invention and the existing porcine thymosin were prepared into solutions containing 1mg of polypeptide per 1ml, respectively, as test solutions using Hank's solution.

(4) Measuring 6 test tubes, adding Hank's solution 0.1ml into each of 3 test tubes as control tube, adding test solution 0.1ml into each of 3 test tubes, adding E receptor-free thymus T cell suspension 0.2ml into each of 3 test tubes, keeping at 37 deg.C for 1 hr, adding sheep erythrocyte suspension 0.2ml, shaking, centrifuging at 500 rpm for 3min, placing in 4 deg.C refrigerator overnight, taking out the next day, discarding supernatant, adding one drop of stationary liquid into each tube, shaking gently, standing for 10min, adding 2 drops of staining solution, shaking, standing for 15 min, counting the number of blue cells on microscope, counting the number of lymphocytes (not less than 200) on 16 large squares, counting the number of cells formed by E roses (combining with lymphocytes of more than 3) to obtain percentage of E roses, and (6) taking an average value. The average value of the test tube or the control tube is obtained.

Sample viability assay tube E rosette percentage-control tube E rosette percentage

3. Results

The results show that the activity of the porcine thymosin de-E receptor method prepared in the embodiment 1 of the invention is obviously higher than that of the existing porcine thymosin, and is respectively 18.7% and 13.2%, as shown in figure 1. The result shows that the pig thymosin in the embodiment 1 of the invention can obviously improve the function of the T cells of the thymus to recover the E receptor thereof and enhance the immunologic function of the organism, and the effect is obviously superior to that of the existing pig thymosin.

Comparative example 2

Experiment for comparing influence of porcine thymosin prepared in example 2 of the invention and existing porcine thymosin on mouse spleen lymphocyte proliferation

In the experiment, the influence of the porcine thymosin on the mouse spleen lymphocyte proliferation activity is detected by adopting a 4-Methyl azo blue Method (MTT), so that the enhancement effect of the porcine thymosin on the organism immune function is reflected, and the biological activity conditions of the porcine thymosin and the existing porcine thymosin in the embodiment 2 of the invention are known.

1 test Material

1.1 healthy Kunming mice weighing 18.0 g. + -. 2.0 g.

1.2 reagents potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium bicarbonate, citric acid, sodium citrate, glucose, lymphocyte separation medium, RPMI-1640 culture medium, penicillin streptomycin solution-double antibody, newborn bovine serum, MTT, dimethyl sulfoxide (DMSO).

1.3 instrumental centrifuge, optical microscope, CO ₂ Incubator and enzymeAnd (4) marking the instrument.

2 test method

2.1 preparation of the solution

2.1.1 preparation of sample solution to be tested the porcine thymosin peptide of the invention example 2 and the existing porcine thymosin peptide were each diluted with physiological saline in a gradient manner to make the polypeptide concentrations 1.0mg/ml, 0.75mg/ml and 0.5 mg/ml.

2.1.2MTT solution 0.100g of MTT powder was finely weighed, dissolved in 20ml of PBS, and sterilized by filtration through a 0.22 μm filter. Storing at 4 ℃ in the dark.

2.1.3 Hank's solution 0.3% potassium dihydrogen phosphate solution, 0.76% disodium hydrogen phosphate solution, 2% potassium chloride solution and 20% sodium chloride solution are mixed in sequence according to the ratio of 20:20:20:40, 1.0g of glucose is added, the mixture is dissolved and mixed evenly, water is used for diluting the mixture to 1000ml, and 4% sodium bicarbonate solution is used for adjusting the pH value to 7.2-7.3 (prepared in the near-use process).

2.1.4 measurement of maintenance culture fluid newborn bovine serum 2ml, adding RPMI 1640 culture fluid to 100ml, and storing at 4 deg.C for use.

2.2 preparation of mouse spleen lymphocyte suspension 5 mice were killed by cervical dislocation, sterilized and then placed on an aseptic console, the spleen was aseptically taken and placed in a mortar and an appropriate amount of Hank's solution was added for grinding, after 100 mesh filtration, the cells were washed with an appropriate amount of Hank's solution, centrifuged at 1500r/min for 3-5 min, the supernatant was discarded, and the washing was repeated three times (same operation as before). Resuspending the cells in appropriate amounts of Hank's solution and adding to the centrifuge tube containing 1/3 volumes of the separated medium, centrifuging at 2000r/min for 20min, carefully aspirating the spleen lymphocytes in the middle layer and washing 3 times (same procedure as above), resuspending the cells in appropriate amounts of maintenance medium and diluting the cells to a final concentration of 1.0X 10 per 1.0ml ⁶ And (4) cells.

2.3 cell culture A suspension of mouse spleen lymphocytes was added to a 96-well plate at 100. mu.l/well for 50 wells at 37 ℃ with 5% CO ₂ Preculture was carried out under the conditions.

2.4 test grouping mouse spleen lymphocytes are pre-cultured for 4-6 h and then divided into an experimental group 1(15 holes/group), an experimental group 2(15 holes/group) and a blank group (5 holes/group), wherein the experimental group 1 is added with the pig thymosin of the invention embodiment 2, the experimental group 2 is added with the existing pig thymosin, the blank group is added with physiological saline and then continuously cultured (as shown in table 1), and the details are as follows:

experimental group 1: adding the solution of the porcine thymosin to be detected in the embodiment 2 of the invention with polypeptide concentrations of 1.0mg/ml, 0.75mg/ml and 0.5mg/ml into the test holes of the experimental group 1, wherein 5 holes are added for each concentration, and 20 mul is added for each hole;

experimental group 2: respectively taking the existing solutions of the porcine thymosin to be detected with the polypeptide concentrations of 1.0mg/ml, 0.75mg/ml and 0.5mg/ml, adding the solutions into test holes of the experimental group 2, adding 5 holes for each concentration, and adding 20 mul for each hole;

blank group: physiological saline was added to the test wells of the blank set at 20. mu.l/well.

TABLE 1 design of the experiments

2.5 assay after 48h incubation, 10. mu.l of MTT solution was added to each well, and 5% CO was added at 37 ℃ ₂ Culturing for 4h under the condition, carefully discarding the supernatant, adding 100 μ l of dimethyl sulfoxide into each well, oscillating for 10min, placing into an enzyme-labeling instrument, measuring absorbance with 630nm as reference wavelength and 550nm as measurement wavelength, and recording OD _550nm The value is obtained.

2.6 data processing to calculate Each set of OD _550nm Mean, and the resulting data were analyzed by one-way analysis of variance and Least Significant Difference (LSD), P<A difference of 0.05 was significant.

3 results of

The absorbance values at 550nm for each well are shown in Table 2. At polypeptide concentrations of 1.0mg/ml, 0.75mg/ml and 0.5mg/ml, the absorbance values for both experimental 1 and experimental 2 were significantly higher than the blank (P <0.05) and the absorbance value for experimental 1 was significantly higher than the absorbance value for experimental 2(P < 0.05). In addition, the polypeptide concentration of 0.75mg/ml absorbance value is higher than other 2 concentrations. The result shows that the pig thymosin in the embodiment 2 can obviously promote the proliferation of mouse spleen lymphocytes and enhance the immune function of an organism, and the effect is obviously superior to that of the existing pig thymosin.

TABLE 2 Effect of porcine thymosin on mouse spleen lymphocyte proliferation (OD) _550nm )

Note: the lower case letters with different superscripts indicate significant differences (P < 0.05).

Comparative example 3

Exogenous virus test comparison experiment is carried out on the porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin

1 test Material

1.1 Virus detection kit bovine viral diarrhea virus indirect immunofluorescence detection kit, porcine parvovirus indirect immunofluorescence detection kit, classical swine fever virus indirect immunofluorescence detection kit, porcine circovirus type 2 indirect immunofluorescence detection kit, porcine reproductive and respiratory syndrome virus universal RT-PCR detection kit, porcine pseudorabies virus PCR detection kit, and foot and mouth disease virus multiplex RT-PCR detection kit.

1.2 strains of bovine viral diarrhea virus (strain NADL), porcine parvovirus (strain NADL-2), hog cholera virus (strain French Thiverval), porcine circovirus type 2 (strain DBN-SX 07).

1.3 cells MDBK cells, ST cells, Vero cells and PK-15 cells.

1.4 reagents of giemsa dye, methanol, acetone, guinea pig red blood cells, chicken red blood cells, African swine fever P72 gene recombinant plasmid, DNA extraction kit, 10 XTaq buffer (containing 25mmol/L Mg) ²⁺ ) Taq DNA Polymerase, dNTP mix (10mM), DNA molecular weight standard DL2000, 50 XTAE electrophoresis buffer.

1.5 Instrument CO ₂ Incubator, inverted fluorescence microscope, PCR instrument.

2 test method

Each of 3 bottles of the porcine thymosin peptide prepared in example 3 of the present invention and the conventional porcine thymosin peptide was mixed and used as a sample for the following experiments.

2.1 bovine viral diarrhea Virus, porcine parvoVirus, and hog cholera Virus testing

The test is carried out according to the appendix of the existing Chinese animal pharmacopoeia, and the test method specifically comprises the following steps:

2.1.1 inoculation and culture of sample the tested sample is inoculated with MDBK cells, ST cells and Vero cells which have grown into good single layers respectively (single layer inoculation), ST cells are synchronously inoculated, 2.0ml of the test sample is inoculated into each bottle, simultaneously MDBK, ST and Vero normal cell controls are set, after the test sample is placed at 37 ℃ for culture for 5 days, freeze thawing is carried out repeatedly for 3 times, and a cell culture is collected (F1); in this way, 2 passages were blind and cell cultures were collected (F2).

The cell culture was observed during the culture period. If the cell lesion appears during the culture period, the cell lesion is judged not to be in accordance with the regulation; if there is no cytopathic effect, the fluorescent antibody test, cytopathic test and erythrocyte adsorptive exogenous virus test are performed. The method comprises the following specific steps:

2.1.2 fluorescent antibody assay

2.1.2.1 selection of cells bovine viral diarrhea virus was tested with MDBK cells (monolayer inoculation); porcine parvovirus was examined with ST cells (synchronized vaccination); hog cholera virus was tested on ST cells (monolayer inoculation).

2.1.2.2 cell plate inoculation the cell culture prepared in 2.1.1 (F2) was inoculated with 96-well MDBK cell plates and ST cell plates, respectively, which had grown into good monolayers (monolayer inoculation), and simultaneously inoculated with ST cell plates, 100. mu.l per well. At the same time, the FAIDs are respectively set at 100-300 ₅₀ Bovine viral diarrhea virus (single layer inoculation), classical swine fever virus (single layer inoculation), and porcine parvovirus (simultaneous inoculation) were used as positive controls, and normal cell controls were established. Placing the cell culture, positive control and normal cell control of the sample at 37 deg.C and 5% CO ₂ And (5) culturing in an incubator for 5 d.

2.1.2.3 Virus test

Referring to the instructions of the indirect immunofluorescence detection kit for bovine viral diarrhea virus, porcine parvovirus and classical swine fever virus, the primary antibody and the secondary antibody corresponding to each kit are respectively adopted for dyeing, and the specific steps are as follows:

2.1.2.3.1 the cultured cell monolayer in 2.1.2.2 was taken, the culture solution was discarded, and the cell was washed 1-2 times with PBS. And adding 200 mu l of PBS into each hole during each washing, incubating at room temperature for 3-5 min, and discarding the PBS.

2.1.2.3.2 adding 100 μ l of 80% cold acetone solution into each hole of the cell plate, fixing at 2-8 deg.C for 30min, removing acetone, and air drying.

2.1.2.3.3 the cell surface was washed 1 time with PBS and 50. mu.l of primary antibody diluted appropriately with PBS was added to each well and allowed to wet-box at 37 ℃ for 1.0 h.

2.1.2.3.4 repeat 2.1.2.3.1.

2.1.2.3.5 the wash solution was discarded and secondary antibody diluted appropriately with PBS was added at 50. mu.l/well and incubated at 37 ℃ for 1.0h in a wet box.

2.1.2.3.6 repeat 2.1.2.3.1.

2.1.2.3.7 was observed under an inverted microscope at 100X to 200X with blue excitation light (wavelength 490 nm).

2.1.2.3.8 decision

The test is established when the positive control wells show typical specific yellow-green fluorescence and the negative control wells show no specific yellow-green fluorescence. Otherwise, judging that the test has no result.

If the specific yellow-green fluorescence appears in the cell hole to be detected, the cell hole is judged to be positive. Otherwise, the result is judged to be negative.

2.1.3 cytopathic test and erythrocyte-adsorbing foreign virus test

2.1.3.1 cell monolayer culture

The cell culture (F2) prepared in 2.1.1 was inoculated into 100. mu.l of each well-monolayer ST cell plate and Vero cell plate, and the plates were cultured in a 37 ℃ incubator for 7 days.

2.1.3.2 cytopathic assay

Removing culture solution from the cultured cell monolayer of 2.1.3.1, washing with PBS for 1 time, adding 80% acetone, fixing for 10min, naturally drying, adding 0.1ml of dyeing solution of Giemsa, dyeing for 30min at room temperature, washing with PBS for 3 times, inspecting the cell monolayer with microscope, and observing whether inclusion body, abnormal cell number and other cytopathic effect caused by virus exist. The inoculated monolayer cells were judged to be defective when compared with the cell control, if specific CPE was found due to the exogenous virus. If the possibility of contamination by the foreign virus is suspected, the foreign virus is judged to be unqualified if the possibility cannot be excluded by other tests.

2.1.3.3 erythrocyte adsorptive exogenous virus test method

The cultured cell monolayer of 2.1.3.1 was discarded, washed 3 times with PBS, 0.1ml of an equal amount of a mixture of guinea pig erythrocytes and chicken erythrocytes of 0.2% was added to each cell well, the plate was left at 2 to 8 ℃ and 20 to 25 ℃ for 30min at the same time, then washed 3 times with PBS, and the adsorption was examined by a 100X microscope. If specific erythrocyte adsorption by exogenous viruses is found, the cells are judged to be unqualified. If the possibility of contamination by the foreign virus is suspected, the foreign virus is judged to be unqualified if the possibility cannot be excluded by other tests.

2.2 porcine reproductive and respiratory syndrome Virus test

The test was carried out according to the "epiboly exogenous virus test method".

2.3 foot and mouth disease Virus test

2.4 Pseudorabies Virus test

2.5 porcine circovirus type 2 test

The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia.

2.6 African Swine fever Virus test

The test was carried out according to the "ordinary PCR detection method" in the appendix of No. 172 < method for detecting nucleic acid of African swine fever virus in biological products for pigs and related swine-derived raw and auxiliary materials, published by the Ministry of agricultural rural areas.

3 results

3.1 bovine viral diarrhea Virus, porcine parvovirus, classical Swine fever Virus test results

3.1.1 fluorescent antibody assay

The porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin are sequentially subjected to bovine viral diarrhea virus, porcine parvovirus and classical swine fever virus detection by adopting a fluorescent antibody detection method.

In the cell culture period, the observation result of the cell culture shows that the F1 generation cells and the F2 generation cells inoculated with the porcine thymosin prepared by the invention in example 3 and the existing porcine thymosin grow normally, and abnormal rounding, vacuole, aggregation, particle increase, atrophy, shedding and the like do not occur.

The result of the fluorescent antibody examination shows that the positive control holes all show typical specific yellow-green fluorescence, the negative control holes do not show specific yellow-green fluorescence, and the test is established. In the test of bovine viral diarrhea virus and porcine parvovirus, the cell holes to be tested inoculated with the porcine thymosin and the conventional porcine thymosin in the embodiment 3 of the invention have no specific yellow-green fluorescence, and are judged to be negative. In the hog cholera virus test, the porcine thymosin of the embodiment 3 of the invention is inoculated without specific yellow-green fluorescence, and the porcine thymosin is judged to be negative; and the inoculated thymus peptide of the existing pig has typical specific yellow green fluorescence and is judged to be positive.

The results show that the porcine thymosin prepared in the embodiment 3 of the invention has no pollution of bovine viral diarrhea virus, porcine parvovirus and classical swine fever virus. The existing porcine thymosin has no pollution of bovine viral diarrhea virus and porcine parvovirus, but has the pollution of classical swine fever virus.

3.1.2 cytopathic test and test results for erythrocyte-adsorbing exogenous Virus

Exogenous virus detection is carried out on the pig thymosin and the existing pig thymosin prepared in the embodiment 3 of the invention by adopting a cytopathogenic detection method and an erythrocyte adsorption exogenous virus detection method, and detection results show that the pig thymosin and the existing pig thymosin to be detected do not have specific CPE caused by exogenous viruses and do not have erythrocyte adsorption phenomenon caused by exogenous viruses.

The results show that the results of the cytopathogenic test and the erythrocyte adsorptive exogenous virus test of the porcine thymosin prepared in the embodiment 3 of the invention and the conventional porcine thymosin are qualified.

3.2 porcine reproductive and respiratory syndrome Virus test results

The porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin are sequentially subjected to porcine reproductive and respiratory syndrome virus test by an RT-PCR method, and test results show that 436bp amplification bands appear in positive controls of the porcine reproductive and respiratory syndrome virus, no amplification band appears in negative controls, and an experimental result is established; the pig thymosin prepared in the embodiment 3 of the invention and the existing pig thymosin to-be-detected sample have no 436bp amplification band, and are judged to be negative.

The result shows that the porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin have no viral pollution of porcine reproductive and respiratory syndrome.

3.3 foot-and-mouth disease Virus test results

The pig thymosin prepared in the embodiment 3 of the invention and the existing pig thymosin are subjected to foot-and-mouth disease virus detection in sequence by adopting an RT-PCR method, and the detection result shows that 634bp, 483bp and 278bp amplification bands appear in positive controls of the foot-and-mouth disease virus, no amplification band appears in negative controls, and the experimental result is established; neither the porcine thymosin prepared in the embodiment 3 of the invention nor the existing sample to be detected of the porcine thymosin has the amplified bands of 634bp, 483bp and 278bp, and the sample is judged to be negative.

The result shows that the pig thymosin prepared in the embodiment 3 of the invention and the existing pig thymosin have no foot and mouth disease virus pollution.

3.4 Pseudorabies Virus test results

Adopting a PCR method to carry out pseudorabies virus detection on the porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin in sequence, wherein the detection result shows that 217bp amplification bands appear in the positive control of the pseudorabies virus, no amplification band appears in the negative control, and the experimental result is established; the porcine thymosin prepared in the embodiment 3 of the invention and the existing sample to be detected of the porcine thymosin have no 217bp amplification band, and are judged as negative tests.

The results show that the porcine thymosin prepared in the embodiment 3 of the invention and the existing porcine thymosin have no pseudorabies virus pollution.

3.5 porcine circovirus type 2 test results

Porcine circovirus type 2 testing is performed on the porcine thymosin peptide prepared in the embodiment 3 of the invention and the existing porcine thymosin peptide by a fluorescent antibody detection method.

The result of the fluorescent antibody examination shows that the positive control holes all have typical specific intracellular yellow-green fluorescence, and the negative control holes have no specific yellow-green fluorescence, so that the test is established; the cell holes to be detected inoculated with the porcine thymosin and the conventional porcine thymosin in the embodiment 3 of the invention have no specific yellow-green fluorescence in nucleus and are judged to be negative.

The result shows that the pig thymosin prepared in the embodiment 3 of the invention and the existing pig thymosin have no pollution of the porcine circovirus type 2.

3.6 African Swine fever Virus test results

The pig thymosin and the existing pig thymosin in the embodiment 3 of the invention are subjected to African swine fever virus test by adopting a PCR method, and test results show that a positive control shows a 257bp specific band, a negative control does not show a 257bp specific band, and the test is established; in the embodiment 3 of the invention, no amplification band with the size consistent with that of the positive control appears in the pig thymosin and the existing pig thymosin, and the pig thymosin is judged to be negative to the African swine fever virus nucleic acid.

The result shows that the pig thymosin and the existing pig thymosin in the embodiment 3 of the invention are free from the pollution of the African swine fever virus.

Extrinsic injection-exogenous virus inspection method

1 sample treatment

1.1 processing raw material pig thymus gland samples, wherein the number of randomly extracted pig thymus gland samples is not less than 1% of the total number of the whole pig thymus gland, each pig thymus gland sample is about 3.0g, the pig thymus glands are mixed and homogenized, the pig thymus gland samples are repeatedly frozen and thawed and then centrifuged, and the centrifuged supernatant is taken and sterilized by a 0.22 mu m sterilization filter for later use.

1.2 processing the porcine thymosin sample, taking 3 bottles of the same batch of the porcine thymosin, mixing, and taking the mixture as a sample to be detected.

2 bovine viral diarrhea Virus, hog cholera Virus and porcine circovirus type 2 test

The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the test results are negative.

3 porcine parvovirus assay

The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the test result is negative.

3.1 cell culture preparation

3.1.1 Simultaneous inoculation of the sample with ST or PK-15 cell suspensions into cell culture flasks at 5% CO ₂ Culturing at 37 ℃ for 3-5 days, repeatedly freezing and thawing for 3 times, collecting cell culture, and performing blind passage for 3 generations by the method.

3.1.2 synchronously inoculating the cell culture to be detected and the ST or PK-15 cell suspension into a 96-well cell culture plate, wherein each well has 100 mu l of cell culture, each sample has 24 wells, and a porcine parvovirus positive control and a normal cell control are simultaneously established in each 24 wells in 5% CO ₂ And culturing at 37 ℃ for 3-5 days.

3.2 Virus testing

3.2.1 taking the cultured cell monolayer in the 3.1.2, discarding the culture solution, and washing with PBS for 1-2 times. And adding 200 mu l of PBS into each hole during each washing, incubating at room temperature for 3-5 min, and discarding the PBS.

3.2.2 adding 100 mul of 80% cold acetone solution into each hole, fixing for 30min at 2-8 ℃, then removing the acetone, and naturally drying.

3.2.3 Wash the cell surface 1 time with PBS, then add 50. mu.l of porcine parvovirus-specific monoclonal antibody (primary antibody) per well diluted appropriately with PBS, wet-box at 37 ℃ for 1.0 h.

3.2.4 repeat step 3.2.1.

3.2.5 the wash solution was discarded and FITC-labeled rabbit anti-porcine IgG (secondary antibody) diluted appropriately with PBS was added at 50. mu.l/well and wet-box incubated at 37 ℃ for 1.0 h.

3.2.6 repeat step 3.2.1.

3.2.7 Observation under an inverted microscope at 100X to 200X with blue excitation light (wavelength 490nm) magnification.

3.3 determination of results

The test is established when the positive control shows typical specific intracellular yellow-green fluorescence, and the normal cells do not show specific yellow-green fluorescence. If the specific yellow-green fluorescence in the nucleus appears in the cell hole of the sample to be detected, the cell hole is judged to be positive, and the result is not in accordance with the regulation. Otherwise, the result is negative, namely the result meets the regulation. If the positive control does not show specific fluorescence or normal cells show specific fluorescence, no result is judged, and the detection is required to be carried out again.

4 porcine reproductive and respiratory syndrome Virus test

The general RT-PCR detection kit for the porcine reproductive and respiratory syndrome virus of Beijing Shijiheng animal epidemic prevention technology Limited company is used for detection, and the detection result is negative.

4.1 extraction of viral RNA

4.1.1 mu l of each sample, positive control and negative control are respectively added with 600 mu l of lysis solution, fully inverted and mixed evenly, and kept stand for 3-5 min at room temperature.

4.1.2 absorbing the liquid into the adsorption column (the adsorption column is sleeved with a collecting pipe, suspended impurities are not absorbed as much as possible when the liquid is absorbed, so that the adsorption column is prevented from being blocked during centrifugation), and centrifuging at 13000r/min for 30 s.

4.1.3 discard the liquid in the collection tube, add 600. mu.l of wash solution, centrifuge for 30s at 13000 r/min.

4.1.4 repeat step 4.1.3.

4.1.5 discard the liquid in the collection tube, 13000r/min empty column centrifugation for 2min to remove the residual washing liquid.

4.1.6 transferring the adsorption column into a new 1.5ml centrifuge tube, adding 50 μ l of eluent into the center of the column, standing at room temperature for 1min, centrifuging at 13000r/min for 30s, and obtaining the template RNA as the liquid in the centrifuge tube.

4.2 RT-PCR amplification

4.2.1 Total volume 20. mu.l each, containing 16.8. mu.l RT-PCR reaction (mix well before use), 1.2. mu.l enzyme mix, 2. mu.l template RNA.

4.2.2 the following procedure was performed in a PCR amplificator: (1)45 min at 42 ℃ and 3min at 95 ℃. (2) Circulating for 35 times at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 30 s; extension was carried out for 7min at 72 ℃.

4.3 analysis and determination of results

After electrophoresis is finished, the gel plate is taken out and placed on an ultraviolet transilluminator, an ultraviolet lamp is turned on for observation, and when a 436bp amplification band appears in a positive control and no band appears in a negative control (except a primer band), the test result is established. The 436bp amplification zone of the sample to be detected is positive for the porcine reproductive and respiratory syndrome virus, otherwise, the amplification zone is negative.

5 foot and mouth disease Virus test

The test is carried out according to a foot-and-mouth disease virus multiplex RT-PCR detection kit of Lanzhou veterinary research institute of Chinese academy of agricultural sciences, and the test result is negative.

5.1 extraction of viral RNA

5.1.1 Add 560. mu.l of Carrier RNA working solution to a clean 1.5ml centrifuge tube using a pipette.

5.1.2 Add 140. mu.l sample to the centrifuge tube (the sample is allowed to equilibrate to room temperature). Vortex and shake for 15s to mix, in order to ensure that the lysis is sufficient, the sample and Carrier RNA working solution need to be thoroughly mixed.

5.1.3 incubation at room temperature (15-25 ℃) for 10 min.

5.1.4 brief centrifugation to collect liquid attached to the tube walls and tube caps.

5.1.5 Add 560. mu.l of absolute ethanol and vortex on the tube lid for 15 s.

5.1.6 brief centrifugation to collect liquid attached to the tube walls and tube caps.

5.1.7 carefully transfer 630. mu.l of the liquid from the centrifuge tube to RNase-Free adsorption column CR2 (the adsorption column is placed in the collection tube), cover the tube, centrifuge at 8000r/min for 1min, discard the waste liquid, and place the adsorption column back into the collection tube.

5.1.8 the remaining centrifuge tubes were again passed through the column as in step 5.1.7.

5.1.9 carefully open the adsorption column cover, add 500. mu.l solution GD (check whether add absolute ethanol before use), cover the tube cover, centrifuge at 8000r/min for 1min, discard the waste liquid, put the adsorption column back into the collection tube.

5.1.10 carefully open the cover of the adsorption column, add 500 μ l solution RW (check whether absolute ethanol is added before use), cover the tube cover, centrifuge at 8000r/min for 1min, discard the waste liquid, and place the adsorption column back into the collection tube.

5.1.11 repeat step 5.1.10.

5.1.12 the adsorption column is put back into the collection tube, centrifuged at 12000 r/min for 3min to dry the adsorption membrane completely, and the waste liquid is discarded. The remaining ethanol may have an effect on subsequent tests.

5.1.13 the adsorption column was put back into the 2.0ml collection tube, the lid of the adsorption column was opened, and the column was left at room temperature for 3min to dry the adsorption film completely.

5.1.14 the adsorption column is placed in an RNase-Free centrifuge tube (1.5ml), the cover of the adsorption column is carefully opened, and 60 μ l of RNase-Free ddH is suspended and dropped into the middle part of the adsorption membrane ₂ O, covering the cover, and standing at room temperature for 5 min. Centrifuging at 8000r/min for 1 min.

5.2 RT-PCR

5.2.1 Total volume 25. mu.l each, containing 12.5. mu.l of 2 XMRT-PCR Buffer, 3.0. mu.l of Primer Mix, 1.0. mu.l of mRT-PCR Enzyme Mix, 3.5. mu.l of RNase-free water, 5. mu.l of Total RNA.

5.2.2 the following procedure was performed in a PCR amplificator: (1) 30min at 45 ℃; 94 ℃ for 3 min. (2) Circulating for 35 times at 94 ℃ for 30s, at 58 ℃ for 30s and at 72 ℃ for 30 s; extension was then carried out at 72 ℃ for 5 min.

5.3 result analysis and determination

After electrophoresis is finished, the gel plate is taken out and placed on an ultraviolet transilluminator, an ultraviolet lamp is opened for observation, and the positive control electrophoresis result is 3 bands with different sizes, namely 634bp, 483bp and 278 bp. If at least one DNA band of the sample amplification product is consistent with the above band sizes, and the blank control has no amplification band, the sample is judged to be positive.

6 Pseudorabies Virus test

The detection is carried out according to a PCR detection kit of the porcine pseudorabies virus of Beijing Shijiheng animal epidemic prevention technology Co., Ltd, and the detection result is negative.

6.1 sample treatment samples, positive control and negative control 100. mu.l, respectively adding 200. mu.l of digestion solution and 20. mu.l of proteinase k, shaking, mixing, and digesting in 56 deg.C water bath for 1.0 h.

6.2 extraction of viral DNA

6.2.1 taking out the sample, the positive control tube and the negative control tube from the water bath kettle, cooling to room temperature, adding 300 μ l of DNA binding solution, reversing, mixing, transferring all the solution into an adsorption column (the adsorption column is sleeved with a collecting tube, and the suspension impurities are not absorbed as much as possible when the solution is absorbed so as not to block the adsorption column during centrifugation), standing at room temperature for 3min, and centrifuging at 10000r/min for 30 s.

6.2.2 discard the liquid in the collection tube, add 500. mu.l DNA washing solution, centrifuge for 30s at 10000 r/min.

6.2.3 repeat step 6.2.2.

6.2.4 discard the liquid in the collection tube, 10000r/min empty column centrifugation for 1min to remove the residual DNA washing solution.

6.2.5 putting the adsorption column into a new 1.5ml centrifuge tube, adding 50 μ l DNA eluent (preferably preheated at 56 deg.C) into the center of the column, standing at room temperature for 2min, centrifuging at 10000r/min for 30s, and collecting the liquid in the centrifuge tube as template DNA.

6.3 PCR amplification

6.3.1 Total volume 20. mu.l each, containing 16. mu.l PCR reaction mix (mix well before use), 2. mu.l Taq DNA polymerase, 2. mu.l template DNA.

6.3.2 the following procedure was performed on a PCR amplification machine: (1)94 ℃ for 3 min. (2) Circulating for 35 times at 94 ℃ for 30s, 65 ℃ for 30s and 72 ℃ for 30 s; extension was carried out for 7min at 72 ℃.

6.4 result analysis and determination

After electrophoresis is finished, the gel plate is taken out and placed on an ultraviolet transilluminator, an ultraviolet lamp is turned on for observation, 217bp amplification bands appear in positive control, and when no bands appear in negative control (except primer bands), the test result is established. The 217bp amplification band of the sample to be detected is PRV positive, otherwise, the sample to be detected is negative.

7 African Swine fever Virus test

The test is carried out according to No. 172 of the nucleic acid detection method of African swine fever viruses in biological products for pigs and related pig source raw and auxiliary materials, which is published by the rural area of agriculture, and the test result is negative.

Claims

1. A preparation method of pig thymosin is characterized by comprising the following steps: which comprises the following steps:

2) breaking the homogenate, keeping the temperature for 0.5-2h in water at 60 ℃, and centrifuging to respectively obtain a centrifugal supernatant and a centrifugal precipitate;

4) gradually performing tangential flow filtration on the permeate by using filter membranes with different cut-off pore diameters and molecular weights, and collecting the permeate to obtain the required crude products of the porcine thymosin with different molecular weight ranges;

5) mixing at least one thymosin crude product with a molecular weight range in the step 4) to obtain a pig thymosin crude product;

2. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: in the step 2), mixing the centrifugal precipitate with water for injection or water for injection with pH value adjusted to 1.5-6.5 by hydrochloric acid, homogenizing by a colloid mill to obtain homogenate, carrying out cell disruption, keeping the temperature for 0.5-2h at 60 ℃, centrifuging to obtain centrifugal supernatant, and combining the two centrifugal supernatants.

3. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: the step 4) is as follows: tangential flow filtration is carried out on part of the permeate in the step 3) by adopting a 5-10kDa filter membrane, and the permeate is collected, namely a crude product of the porcine thymosin with the molecular weight of less than 5-10 kDa;

performing tangential flow filtration on part of the permeate in the step 3) by adopting a 10-1000kDa filter membrane, collecting the permeate, performing tangential flow filtration by adopting a 1-3kDa filter membrane, and collecting the permeate, namely the crude product of the porcine thymosin with the molecular weight of less than 1-3 kDa.

4. The method of claim 3, wherein the pig thymosin peptide is prepared by the following steps: and step 5) mixing at least one thymosin crude product with a molecular weight range in the step 4), wherein the volume ratio of the porcine thymosin with the molecular weight of less than 1kDa is 30-70%.

5. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: in the step 1), the pig thymus is polluted by bovine avirus diarrhea virus, porcine parvovirus, hog cholera virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, foot and mouth disease virus, pseudorabies virus or African swine fever virus.

6. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: in step 2), the cell disruption method comprises at least one of the following methods: repeated freeze thawing and crushing; crushing by a high-pressure homogenizer; and crushing by using an ultrasonic cell crusher.

7. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: in step 6), the virus removal/inactivation method includes at least two of the following methods:

a pH incubation method: reacting at 4-25 ℃ for 2h-1d when the pH is 2.0-4.0;

② a membrane filtration method: filtering with 15-45nm virus-removing filter;

③ the inactivation method of beta-propiolactone: inactivating at 0.001% -0.025% concentration at 4 deg.C for 6-24 hr, and hydrolyzing at 37 deg.C for 2-8 hr;

and fourthly, formaldehyde inactivation: inactivating at 37 deg.C for 6-12h at 0.01% -0.05%;

pasteurization: inactivating at 60 deg.C for 3-5 h.

8. The method of claim 1, wherein the step of preparing the porcine thymosin peptide comprises the steps of: the porcine thymosin obtained in the step 6) is free from pollution of bovine viral diarrhea virus, swine fever virus, porcine parvovirus, porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus or African swine fever virus.

9. The use of the porcine thymosin peptide obtained by the preparation method of claim 4 for increasing the activity of animal lymphocytes.