A polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome and its application

Abstract

The present invention belongs to the field of biological medicine technology and discloses a polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome. The polypeptide composition includes thymosin and spirulina polypeptide, and the mass ratio of thymosin to spirulina polypeptide is (1 - 5):(1 - 3). The polypeptide composition of the present invention has a significant therapeutic effect on porcine reproductive and respiratory syndrome, is safe, has no toxic and side effects, and has no drug residues after use. In addition, the raw materials of the polypeptide composition of the present invention have sufficient market supply and low prices, and have good market application and promotion value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome (PRRS) and its application. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as swine blue ear disease, is caused by a RNA virus which is highly mutable and recombinogenic. As a result, there are many clinically prevalent strains with slightly different clinical symptoms. According to the virulence, PRRS virus can be divided into classical strains, highly pathogenic strains and recombinant variant strains. The clinical manifestations of various strains are as follows: in sows, it mainly shows reproductive disorders, fever, anorexia, abortion, stillbirth, mummified fetus, return to estrus, agalactia, etc.; in piglets, nursery pigs and finishing pigs, it mainly shows respiratory disorders, persistent fever, reduced feed intake, emaciation, rough hair, red ears, interstitial pneumonia, cough, wheezing and other respiratory symptoms. Among them, the recombinant strain NADC30 can also cause diarrhea in young pig populations in addition to the clinical manifestations of other strains, while other strains do not have this symptom of diarrhea. The prevalence rate of NADC30-like strains in China is 55% - 65%, and they are gradually becoming the main prevalent strains, attracting high attention in the industry. At present, the actual effects of using vaccines for prevention and drugs for treatment of PRRS are not ideal.

[0003] Thymosin is a group of small molecular active peptides secreted by thymic tissue epithelial cells, which can be used to treat various diseases and regulate the body's immune function by affecting the activities of lymphocytes and factors such as IL-2 and NK cells. The secretion and activity of thymosin in the body change with age, light, hormone and trace element levels. In recent years, calf thymosin, as an immunomodulator, has been widely used in medical clinics and has also achieved satisfactory results in the prevention and treatment research of veterinary clinics.

[0004] Spirulina is one of the most common natural microalgae, rich in protein, essential amino acids, vitamins and various trace elements, and also has the function of significantly enhancing the body's immune function. In recent years, it has become a common health food for humans. There have been many studies on the active ingredients such as polysaccharides, proteins and glycolipids contained in spirulina. At the same time, the natural active polypeptides in spirulina also have various physiological activities. For example, it has been confirmed that spirulina peptidoglycan can significantly enhance human immunity. However, the research on the functions and applications of spirulina polypeptides in veterinary clinics is still relatively scarce.

[0005] Therefore, it is of great significance to provide a method that can effectively combine the immunomodulatory functions of thymosin and spirulina to prevent and treat PRRS. Summary of the Invention

[0006] Aiming at the problems and deficiencies in the existing technology, the present invention aims to provide a polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome and its application.

[0007] Based on the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a polypeptide composition, comprising thymosin and spirulina polypeptide.

[0009] Preferably, the mass ratio of the thymosin to the spirulina polypeptide is (1-5):(1-3).

[0010] Preferably, the components of the polypeptide composition further include water.

[0011] More preferably, the water is purified water.

[0012] In the second aspect, the present invention provides a preparation method of a polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome. Mix the thymosin and the spirulina polypeptide evenly according to the mass ratio to obtain the polypeptide composition.

[0013] Preferably, the preparation method of the polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome specifically includes the following steps:

[0014] (1) Add water to calf thymus and spirulina powder respectively, and perform fragmentation treatment to obtain calf thymus homogenate and spirulina homogenate. Adjust the pH values of the calf thymus homogenate and the spirulina homogenate to acidic, and then add protease to the calf thymus homogenate and the spirulina homogenate respectively for digestion treatment to obtain calf thymus digestive juice and spirulina digestive juice;

[0015] (2) Heat and filter the calf thymus digestive juice and the spirulina digestive juice obtained in step (1) respectively, and concentrate under reduced pressure to obtain calf thymosin extraction solution and spirulina polypeptide extraction solution;

[0016] (3) Lyophilize the calf thymosin extraction solution and the spirulina polypeptide extraction solution obtained in step (2) respectively to obtain calf thymosin and spirulina polypeptide. Mix the calf thymosin and the spirulina polypeptide according to the mass ratio to obtain the polypeptide composition.

[0017] Preferably, when the components of the polypeptide composition include water, its preparation method specifically includes the following steps:

[0018] (1) Add water to calf thymus and spirulina powder respectively, and perform fragmentation treatment to obtain calf thymus homogenate and spirulina homogenate. Adjust the pH values of the calf thymus homogenate and the spirulina homogenate to acidic, and then add protease to the calf thymus homogenate and the spirulina homogenate respectively for digestion treatment to obtain calf thymus digestive juice and spirulina digestive juice;

[0019] (2)Heat and filter the calf thymus digestive solution and spirulina digestive solution obtained in step (1), and concentrate under reduced pressure to obtain a calf thymosin extraction solution and a spirulina polypeptide extraction solution;

[0020] (3)Mix the calf thymosin extraction solution and the spirulina polypeptide extraction solution obtained in step (2) according to the mass ratio of calf thymosin to spirulina polypeptide to obtain a polypeptide composition.

[0021] Preferably, in step (1), the mass ratio of the calf thymus to water is 1:2, and the mass ratio of the spirulina powder to water is 1:3.

[0022] Preferably, the water in step (1) is purified water.

[0023] Furthermore, in step (1), a flash extraction device is used for the crushing treatment.

[0024] Preferably, the protease in step (1) is an acidic protease, and the dosage of the protease is not less than 600,000 IU per 1 kg of the calf thymus homogenate or spirulina homogenate.

[0025] Furthermore, in step (1), the temperature of the digestion treatment is 45 - 50 °C, and the duration is 4 - 6 h.

[0026] Furthermore, in step (2), the temperature of the heating treatment is 80 - 90 °C, and the duration is 15 - 30 min.

[0027] Furthermore, the specific operation of the filtration treatment in step (2) is as follows: respectively perform microfiltration on the calf thymus digestive solution and the spirulina digestive solution, and then perform ultrafiltration. The ultrafiltration membrane with a molecular weight cut-off of 8000 daltons is used for the ultrafiltration treatment.

[0028] Furthermore, in step (2), the temperature of the concentration under reduced pressure is 55 - 65 °C.

[0029] Preferably, the concentration of the calf thymosin extraction solution in step (2) is 30 mg / mL, and the concentration of the spirulina polypeptide extraction solution is 30 mg / mL.

[0030] In a third aspect, the present invention provides a drug for preventing and treating porcine reproductive and respiratory syndrome, and the main raw materials include the polypeptide composition and pharmaceutically acceptable excipients.

[0031] Preferably, the drug is in an injectable form.

[0032] More preferably, the injectable form is an injection or a freeze-dried powder injection.

[0033] In a fourth aspect, the present invention provides a preparation method of a drug for preventing and treating porcine reproductive and respiratory syndrome, including the following steps:

[0034] (1) Prepare the polypeptide composition by adding water to form a solution with a concentration of 20 mg / ml and adjust the pH value to 6.0 - 7.0.

[0035] (2) Filter and sterilize the polypeptide composition solution obtained in step (1) to obtain a drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), and this drug is an injection.

[0036] Preferably, the water in step (1) is purified water.

[0037] Further, in step (2), a 0.1 - micron filter element is used for the filtration treatment.

[0038] Further, in step (2), the temperature of the sterilization treatment is 105°C and the duration is 30 min.

[0039] In the fifth aspect, the present invention provides the application of the polypeptide composition in the preparation of a drug or pharmaceutical preparation for preventing and treating porcine reproductive and respiratory syndrome (PRRS).

[0040] In the sixth aspect, the present invention provides the application of the drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS) in a pharmaceutical preparation for preventing and treating porcine reproductive and respiratory syndrome (PRRS).

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1. The present invention uses calf thymus and spirulina as raw materials, extracts thymosin and spirulina polypeptide respectively, and prepares a composition of calf thymosin and spirulina polypeptide by mixing them in proportion. For the first time, the composition of the two polypeptides is applied to the treatment of porcine reproductive and respiratory syndrome (PRRS).

[0043] 2. Both the calf thymosin and spirulina polypeptide used in the present invention can effectively stimulate the immune system and significantly improve the immunity of pigs. The combined application of the two plays a reticular activation role of the bioactive polypeptide in the composition on the immune system. The clinical experimental results show that the thymosin and spirulina polypeptide composition prepared by the present invention has a significant therapeutic effect on porcine reproductive and respiratory syndrome (PRRS), with an effective rate of 100%, and is superior to the therapeutic effects of thymosin and spirulina polypeptide used alone on porcine reproductive and respiratory syndrome (PRRS).

[0044] 3. The polypeptide composition of the present invention is safe, non - toxic and has no side effects, and there is no drug residue after use. In addition, the raw materials of the polypeptide composition of the present invention have a sufficient market supply and a low price, and have good market application and promotion value. Brief Description of the Drawings

[0045] Figure 1 It is a graph showing the average body temperature change of the pig group from the 1st to the 14th day after different treatment regimens. Detailed Embodiments

[0046] The following examples are only applicable for further elaborating the present invention. It should be noted that all technologies and scientific terms used in the present invention have the same meaning as those in the technical field to which the present invention belongs, unless otherwise specified. For the experimental methods without specific conditions in the following examples, conventional techniques in this technical field are adopted, or the conditions recommended by the manufacturer are followed; for the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0047] To make the objectives, technical solutions and advantages of the present invention clearer and more definite, the present invention will be further described in detail below through examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0048] Example 1

[0049] A polypeptide composition, which is composed of thymosin and spirulina polypeptide; the mass ratio of thymosin to spirulina polypeptide in the polypeptide composition is 1:1.

[0050] The preparation method of the above polypeptide composition includes the following steps:

[0051] (1) Weigh fresh calf thymus and spirulina powder respectively. According to the mass ratio of fresh calf thymus to purified water being 1:2 and the mass ratio of spirulina powder to purified water being 1:3, use a flash extraction device for cell wall breaking treatment to obtain a calf thymus homogenate and a spirulina homogenate. Adjust the pH values of the calf thymus homogenate and the spirulina homogenate to 2.5 - 3.0 with 10% hydrochloric acid solution, and then add acid protease to the calf thymus homogenate and the spirulina homogenate respectively (the protease addition amount in 1 kg of calf thymus homogenate or spirulina homogenate is 600,000 IU), and carry out digestion treatment at 50 °C for 6 hours to obtain a calf thymus digestive juice and a spirulina digestive juice;

[0052] (2) Heat the calf thymus digestive juice and the spirulina digestive juice obtained in step (1) at 80 °C for 30 min respectively, filter to remove the denatured protein to obtain a crude calf thymosin extract and a crude spirulina polypeptide extract. After microfiltration of the crude calf thymosin extract and the crude spirulina polypeptide extract respectively, carry out ultrafiltration treatment with an ultrafiltration membrane with a molecular weight cut-off of 8000 Da, and then carry out vacuum concentration at 60 °C to obtain a refined calf thymosin extract and a refined spirulina polypeptide extract. The concentration of the refined calf thymosin extract is 30 mg / mL, and the concentration of the refined spirulina polypeptide extract is 30 mg / mL;

[0053] (3) Freeze-dry the refined calf thymosin extract and the refined spirulina polypeptide extract obtained in step (2) respectively to obtain calf thymosin and spirulina polypeptide. Mix the calf thymosin and the spirulina polypeptide according to the mass ratio of 1:1 to obtain the polypeptide composition.

[0054] Example 2

[0055] A polypeptide composition, which is composed of thymosin, spirulina polypeptide and purified water; the mass ratio of thymosin to spirulina polypeptide in the polypeptide composition is 1:1.

[0056] The preparation method of the above polypeptide composition is basically the same as that of Example 1, and the difference is that in step (3), the calf thymosin refined extract and the spirulina polypeptide refined extract are mixed evenly according to the mass ratio of thymosin to spirulina polypeptide of 1:1, and the polypeptide composition is obtained.

[0057] Example 3

[0058] A polypeptide composition, which is basically the same as the content of Example 2, and the difference is that the mass ratio of thymosin to spirulina polypeptide in the polypeptide composition is 3:2.

[0059] The preparation method of the above polypeptide composition is the same as that of Example 2.

[0060] Example 4

[0061] A polypeptide composition, which is basically the same as the content of Example 2, and the difference is that the mass ratio of thymosin to spirulina polypeptide in the polypeptide composition is 3:1.

[0062] The preparation method of the above polypeptide composition is the same as that of Example 2.

[0063] Example 5

[0064] A drug for preventing and treating porcine reproductive and respiratory syndrome is prepared from the polypeptide composition prepared in Example 2 and pharmaceutically acceptable excipients.

[0065] The preparation method of the above drug for preventing and treating porcine reproductive and respiratory syndrome includes the following steps:

[0066] (1) The polypeptide composition is formulated with purified water into a polypeptide composition solution with a concentration of 20 mg / mL and the pH value is adjusted to 6.5.

[0067] (2) After passing the polypeptide composition solution obtained in step (1) through a 0.1-micron filter element to remove impurities, it is sterilized at 105 °C for 30 minutes, and the drug for preventing and treating porcine reproductive and respiratory syndrome is obtained, and this drug is an injection.

[0068] Example 6

[0069] A drug for preventing and treating porcine reproductive and respiratory syndrome is prepared from the polypeptide composition prepared in Example 3 and pharmaceutically acceptable excipients. The preparation method of the above drug for preventing and treating porcine reproductive and respiratory syndrome is the same as that of Example 5.

[0070] Example 7

[0071] A drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS) is prepared from the polypeptide composition prepared in Example 4 and pharmaceutically acceptable excipients. The preparation method of the above drug for preventing and treating PRRS is the same as that in Example 5.

[0072] Control Example 1

[0073] A drug for preventing and treating PRRS is prepared from thymosin and pharmaceutically acceptable excipients.

[0074] The preparation method of the above drug for preventing and treating PRRS includes the following steps:

[0075] (1) Weigh fresh calf thymus. According to the mass ratio of fresh calf thymus to purified water being 1:2, use a flash extraction device for cell wall breaking treatment to obtain a calf thymus homogenate. Adjust the pH value of the calf thymus homogenate to 2.5 - 3.0 with a 10% hydrochloric acid solution, and then add acid protease (the protease addition amount in 1 kg of calf thymus homogenate is 600,000 IU) to the calf thymus homogenate, and carry out digestion treatment at 45 - 50 °C for 6 hours to obtain a calf thymus digestive solution;

[0076] (2) Heat the calf thymus digestive solution obtained in step (1) at 80 °C for 30 min, filter to remove denatured proteins to obtain a crude calf thymosin extract. After microfiltration of the crude calf thymosin extract, carry out ultrafiltration treatment with an ultrafiltration membrane with a molecular weight cut-off of 8000 daltons, and then carry out reduced pressure concentration at 60 °C to obtain a refined calf thymosin extract, and the concentration of the refined calf thymosin extract is 30 mg / mL;

[0077] (3) Dilute the refined calf thymosin extract obtained in step (2) with purified water to prepare a calf thymosin solution with a concentration of 20 mg / mL and adjust the pH value to 6.5. After passing the calf thymosin solution through a 0.1 - micron filter element to remove impurities, sterilize it at 105 °C for 30 minutes to obtain the drug for preventing and treating PRRS, and this drug is an injection.

[0078] Control Example 2

[0079] A drug for preventing and treating PRRS is prepared from spirulina polypeptide and pharmaceutically acceptable excipients.

[0080] The preparation method of the above drug for preventing and treating PRRS includes the following steps:

[0081] (1)Weigh the Spirulina powder. According to the mass ratio of Spirulina powder to purified water being 1:3, use a flash extraction device for cell wall breaking treatment to obtain a Spirulina homogenate. Adjust the pH value of the Spirulina homogenate to 2.5 - 3.0 with a 10% hydrochloric acid solution. Then add acid protease to the Spirulina homogenate (the protease addition amount is 600,000 IU per 1 kg of Spirulina homogenate), and carry out digestion treatment at 45 - 50 °C for 6 hours to obtain a Spirulina digestion solution;

[0082] (2)Heat the Spirulina digestion solution obtained in step (1) at 80 °C for 30 min, filter to remove the denatured protein to obtain a crude Spirulina polypeptide extract. After microfiltration of the crude Spirulina polypeptide extract, carry out ultrafiltration treatment with an ultrafiltration membrane with a molecular weight cut-off of 8000 Da, and then carry out vacuum concentration at 60 °C to obtain a refined Spirulina polypeptide extract, and the concentration of the refined Spirulina polypeptide extract is 30 mg / mL;

[0083] (3)Dilute the refined Spirulina polypeptide extract obtained in step (2) with purified water to prepare a Spirulina polypeptide solution with a concentration of 20 mg / mL and adjust the pH value to 6.5. After passing the Spirulina polypeptide solution through a 0.1 - micron filter element to remove impurities, sterilize it at 105 °C for 30 minutes to obtain a drug for preventing and treating porcine reproductive and respiratory syndrome (PRRS), and this drug is an injection.

[0084] In order to verify the inhibitory effect of the polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome (PRRS) on the lesions of experimental pigs positive for PRRS, the following three test examples were carried out.

[0085] Test Example 1:

[0086] (1)Experimental objects and grouping

[0087] Before the start of the experiment, 60 four - week - old weaned piglets that were negative for both PRRS antigen and antibody were screened from a certain pig farm in Neixiang, Nanyang, Henan. After artificial infection (the strain is TJM - F92 strain, provided by the Infectious Disease Laboratory of Henan Agricultural University, and the infection dose is 10 3 TCID 50 ) 24 h later, 54 pigs with positive PRRS antigen detected by fluorescence quantitative PCR were selected as experimental pigs. Select 6 independent nursery houses and randomly divide the 54 experimental pigs into 6 groups (9 pigs in each group): Experimental Group 1, Experimental Group 2, Experimental Group 3, Experimental Group 4, Experimental Group 5, and Blank Control Group; then randomly divide the 9 experimental pigs in each group into 3 replicate groups according to the pen, that is, 3 experimental pigs in each replicate group.

[0088] (2)Experimental scheme

[0089] In Experimental Group 1, Experimental Group 2, Experimental Group 3, Experimental Group 4, and Experimental Group 5, each pig was intramuscularly injected with the drugs prepared in Example 5, Example 6, Example 7, Comparative Example 1, and Comparative Example 2 at a dose of 1 mg / kg (body weight) per day for 5 consecutive days; in the blank control group, each pig was intramuscularly injected with 1 mL of normal saline per day for 5 consecutive days. The experimental groups and the blank control group were fed and managed daily according to the same feeding and management mode. The experimental scheme is shown in Table 1.

[0090]

[0091] (3)Experimental indicators and data statistics

[0092] On the 1st, 2nd, 3rd, 5th, 7th, and 14th days of the experiment, the content of porcine reproductive and respiratory syndrome virus (PRRSV) was continuously detected to evaluate the duration of the viremia stage of PRRSV in each experimental group; from the 1st to the 14th day of the experiment, the body temperature of the pig population was continuously monitored to evaluate the duration of hyperthermia in each experimental group; before the experiment, the initial body weight of the experimental pigs in each group was statistically analyzed, and after the end of the 28-day experiment, the final body weight, total feed consumption, and final number of live pigs in each group were statistically analyzed. Then, the average daily gain, feed-to-gain ratio, and mortality rate of the experimental pigs were calculated, and the obtained experimental data were analyzed for differences using spss18.0.

[0093] Among them, for the detection of the content of PRRSV, blood swabs were collected from each experimental pig, RNA was extracted using an RNA extraction kit, and finally, fluorescence quantitative PCR was used for detection (a CT value ≤ 35 was determined as positive, 35 < CT value < 40 was determined as suspicious and retested, and a CT value ≥ 40 or no CT value was determined as negative). The calculation formulas for other parameters are as follows: average daily gain (kg) = (final average weight (kg) - initial average weight (kg)) ÷ 28 (d); feed-to-gain ratio = total feed consumption (kg) / (final body weight (kg) + weight of dead and culled pigs (kg) - initial body weight (kg)); mortality rate (%) = (initial number of individuals - final number of live pigs) / initial number of individuals × 100%. In addition, during the experiment, when an experimental pig died or was culled, the weight of the dead and culled pig needed to be weighed and recorded, and the infection status of PRRS in the dead and culled pig needed to be detected and recorded.

[0094] (3)Experimental results

[0095] The positive rates of PRRS antigen on the 1st, 2nd, 3rd, 5th, 7th, and 14th days of each group are shown in Table 2.

[0096]

[0097] As can be seen from Table 2, all were positive from the onset of self-challenge to 14 days in the blank control group, indicating that the challenge model was successful. In Experimental Groups 1 and 2, the positive rate began to gradually decline from the second day after drug administration after challenge, and all turned negative from the 7th to 14th days; in Experimental Group 3, the effect was slightly slower after challenge, and the positive rate did not start to decline until the third day, and not all had turned negative by the 14th day. The effects of Experimental Groups 4 and 5 were relatively weak. Although the positive rate also gradually declined with the prolongation of the drug administration time, the decline amplitude was small.

[0098] The average body temperature of the pig groups from the 1st to 14th days is as Figure 1 shown. As Figure 1 can be seen, with the increase in the body temperature of the pig group after challenge, showing a fever phenomenon, indicating that the challenge model was successful. In Experimental Groups 1 and 2, the body temperature began to gradually return to normal 3 days after drug administration, showing an outstanding effect, while in Experimental Groups 3, 4, and 5, the effect was slower, and the body temperature gradually returned to normal from the 11th to 13th days after drug administration.

[0099] The daily weight gain and feed-to-gain ratio of each group after PRRS virus challenge are shown in Table 3.

[0100]

[0101] Note: In the data of the same column in the above table, the same letter superscript indicates no significant difference (P > 0.05); no same letter superscript, but adjacent letters indicate significant difference (P < 0.05); neither the same nor adjacent letters indicate extremely significant difference (P < 0.01).

[0102] As can be seen from Table 3, in terms of daily average weight gain, compared with the blank control group, the daily average weight gain of Experimental Groups 1 - 5 was significantly increased, and the difference was extremely significant (p < 0.01); there was a significant difference in daily average weight gain between Experimental Group 1 and Experimental Group 2, Experimental Group 2 and Experimental Group 3, and Experimental Group 4 and Experimental Group 5 (0.01 < p < 0.05); there was no significant difference in daily average weight gain between Experimental Group 3 and Experimental Group 4 (p > 0.05). In terms of feed-to-gain ratio, compared with the blank control group, the feed-to-gain ratio of Experimental Groups 1 - 5 was significantly decreased, and the difference was extremely significant (p < 0.01), and there was no significant difference in feed-to-gain ratio between Experimental Group 4 and Experimental Group 5 (p > 0.05). There was no significant difference in the data of daily weight gain and feed-to-gain ratio between Experimental Group 1 and Experimental Group 2, but there was a significant difference from the blank control group and Experimental Groups 3, 4, and 5. Among them, the difference degree between Experimental Group 1 and other groups reached an extremely significant level. It shows that the drug treatment effects of Experimental Groups 1 and 2 after challenge were significant. Although the drugs in Experimental Groups 3, 4, and 5 were also effective, the effects were weaker than those of Experimental Groups 1 and 2.

[0103] The mortality rate of each group after PRRS virus challenge is shown in Table 4.

[0104]

[0105] As can be seen from Table 4, the mortality rates of the experimental group 3, experimental group 4 and the blank control group were significantly higher than those of experimental group 1 and experimental group 2. The treatment effect of experimental group 1 was significant after virus challenge, and no death occurred. However, experimental groups 2, 3, 4 and 5 all had deaths to varying degrees, and the mortality rates of each experimental group were lower than 55.56% of the blank control group, indicating that although the other groups were also effective, their effects were not as good as the drug in experimental group 1.

[0106] In summary, the drug ratio in Example 5 has the best effect, that is, when the ratio of calf thymopeptide to spirulina polypeptide is 1:1, the treatment effect on porcine reproductive and respiratory syndrome is the best, which is reflected in low mortality, high daily weight gain, high feed-to-meat ratio, and rapid body temperature recovery. However, single calf thymopeptide or spirulina polypeptide and when their ratio is 3:2 or 3:1 also have certain effects, but the effects are weaker than those in Example 5.

[0107] Test Example 2:

[0108] (1) Experimental objects and grouping

[0109] In a large-scale pig farm in Zhumadian, Henan, the nursery pigs in three consecutive batches began to show fever symptoms at about 35 days of age, and the duration was about 7 to 15 days. The sampling test results were positive for PRRS antigen (confirmed to be field North American wild virus through testing). In October 2022, 400 nursery pigs were selected and randomly divided into two groups: an experimental group and a blank control group, with 200 pigs in each group.

[0110] (2) Experimental scheme

[0111] The experimental group was treated with the drug prepared in Example 5 of the present invention, and each pig was intramuscularly injected at a dose of 1 mg / kg (body weight) per day for 5 consecutive days; the blank control group was intramuscularly injected with 1 mL / head of normal saline every day for 5 consecutive days. The experimental scheme is shown in Table 5.

[0112]

[0113] (3) Experimental indicators and data statistics

[0114] To detect the situation of PRRS antigen, before and after the medication, 30 pigs were randomly selected for sampling with nasal and oral swabs, and the PRRS antigen was detected by fluorescence quantitative PCR method. Among them, according to the test results before medication, the on-site veterinarian comprehensively determined that this case was porcine reproductive and respiratory syndrome.

[0115] To obtain the fever rate data, observe and count the fever situation of the pig group at 35 days of age. A pig is counted as one fever process from the start to the end of the fever. The calculation formula is: fever rate (%) = the number of feverish pig individuals after medication (head) / 200 (head) * 100%.

[0116] (4) Experimental results

[0117] The blue ear antigen test results and fever rates of the experimental group and the blank control group are shown in Table 6.

[0118]

[0119] As can be seen from Table 6, compared with the blank control group, after using the drug for preventing and treating blue ear disease of pigs prepared by the present invention, the positive rate of blue ear antigen in the pig group decreased by 72.7%, while the positive rate in the blank control group not only did not decrease, but increased. Therefore, the drug for preventing and treating blue ear disease of pigs prepared by the present invention can significantly shorten the blue ear viremia period, significantly reduce the positive rate of blue ear antigen, and effectively shorten the duration of fever in nursery pigs.

[0120] Test Example 3:

[0121] (1) Experimental subjects and groups

[0122] In a large-scale pig farm in Neixiang, Henan, multiple batches of piglets in the delivery room at 7 to 11 days of age often suffered from severe diarrhea, and the mortality rate increased in a short period of time. After one by one investigation of swine diseases that may cause diarrhea, such as porcine epidemic diarrhea, transmissible gastroenteritis, deltavirus, rotavirus, Escherichia coli, Salmonella, Clostridium, etc., all were ruled out, and the effect of conventional diarrhea treatment was also general. However, according to the autopsy results, blue ear virus was detected in the lungs, kidneys and intestines. ORF5 gene sequencing determined that it was a NADC30-like strain. In addition to the clinical manifestations of other strains, this strain can also cause diarrhea in young pigs, so the diarrhea batches were selected for inclusion in the experiment. In May 2023, the company's veterinary team was contacted to conduct a treatment trial, and 200 7-day-old delivery room piglets were selected and divided into two groups, the experimental group and the blank control group, with an average of 100 pigs in each group.

[0123] (2) Experimental plan

[0124] The experimental group used the drug prepared in Example 5 of the present invention, and injected it into the head at a dose of 1 mg / kg, once a day, for 3 consecutive days; the blank control group received an intramuscular injection of 1 mL / head of normal saline every day, for 3 consecutive days. The experimental scheme is shown in Table 7.

[0125]

[0126] (3) Experimental indicators and data statistics

[0127] Before the experiment, the number of pigs with diarrhea in each group was counted. After the experiment, the number of pigs with diarrhea and deaths in each group was counted, and then the mortality rate and diarrhea rate of the pigs were calculated.

[0128] (5) Experimental results

[0129] The mortality rate and diarrhea rate of piglets in the experimental group and the blank control group in the delivery room are shown in Table 8.

[0130]

[0131] As can be seen from Table 8, at the end of the treatment experiment, the mortality rate of the experimental group after treatment was 16%. Compared with the mortality rate of 38% in the blank control group, the mortality rate of the experimental group decreased by 22%. The diarrhea rate of the experimental group decreased from 84% to 26.2% before and after treatment, while the diarrhea rate of the blank control group increased from 74% to 80.7%. The research shows that the polypeptide composition prepared by the present invention can effectively reduce the diarrhea rate and mortality rate of pigs with diarrhea caused by the NADC30-like PRRS virus strain.

[0132] In summary, the present invention effectively overcomes the deficiencies in the prior art and has high industrial utilization value. The role of the above embodiments is to illustrate the substantial content of the present invention, but not to limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A polypeptide composition for preventing and treating porcine reproductive and respiratory syndrome, characterized in that, The polypeptide composition is composed of thymosin and spirulina polypeptide; the mass ratio of thymosin to spirulina polypeptide in the polypeptide composition is (1-5):(1-3); The preparation method of the thymosin is as follows: The bovine thymus is broken by adding water to obtain a bovine thymus homogenate, and the bovine thymus homogenate is digested with a protease to obtain a bovine thymus digestive solution; the bovine thymus digestive solution is heated, filtered, and concentrated under reduced pressure to obtain a bovine thymus peptide extract, and the bovine thymus peptide extract is freeze-dried to obtain thymosin; the preparation method of the spirulina polypeptide is as follows: The spirulina powder is broken by adding water to obtain a spirulina homogenate, and the spirulina homogenate is digested with a protease to obtain a spirulina digestive solution; the spirulina digestive solution is heated, filtered, and concentrated under reduced pressure to obtain a spirulina polypeptide extract, and the spirulina polypeptide extract is freeze-dried to obtain spirulina polypeptide; The specific operation of the filtration treatment in the preparation process of thymosin and spirulina polypeptide is as follows: The bovine thymus digestive solution and the spirulina digestive solution are respectively subjected to microfiltration treatment, and then ultrafiltration treatment is carried out. The ultrafiltration membrane with a molecular weight cut-off of 8000 Da is used for the ultrafiltration treatment.

2. A drug for preventing and treating porcine reproductive and respiratory syndrome, characterized in that, The drug comprises the polypeptide composition as claimed in claim 1 and a pharmaceutically acceptable excipient.

3. The medicament for preventing and treating porcine reproductive and respiratory syndrome according to claim 2, characterized in that, The drug is in an injection dosage form.

4. The medicament for preventing and treating porcine reproductive and respiratory syndrome according to claim 3, characterized in that, The injection dosage form is an injection or a freeze-dried powder injection.

5. The medicament for preventing and treating porcine reproductive and respiratory syndrome according to claim 4, characterized in that, The concentration of the polypeptide composition in the injection is 20-40 mg / mL.

6. A method for preparing the medicament for preventing and treating porcine reproductive and respiratory syndrome according to claim 5, characterized in that, The polypeptide composition is formulated into a polypeptide composition solution, the pH value of the polypeptide composition solution is adjusted to 6.0-7.0, and then filtration and sterilization treatment are carried out to obtain the drug for preventing and treating porcine reproductive and respiratory syndrome.

7. Use of the polypeptide composition as claimed in claim 1 in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome.

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