Recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein and application thereof

Abstract

This invention discloses a recombinant lactic acid bacteria composition expressing the antigen protein of H9N2 subtype avian influenza virus and its application, belonging to the field of biopharmaceutical manufacturing technology. The recombinant lactic acid bacteria composition of this invention comprises: recombinant lactic acid bacteria H9-HA expressing the HA protein of H9N2 subtype avian influenza virus and recombinant lactic acid bacteria H9-NP-M1 expressing the NP and M1 proteins of H9N2 subtype avian influenza virus; wherein the viable count of both recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1 in the recombinant lactic acid bacteria composition is greater than or equal to 10. 10 CFU / g. The recombinant lactic acid bacteria composition of the present invention can effectively alleviate immunosuppression, improve the efficacy of traditional inactivated vaccines, and reduce the viral load in the body after infection with H9N2 subtype avian influenza virus, showing good application prospects in the prevention and control of avian influenza.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical manufacturing, and in particular to a recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein and an application thereof. Background Art

[0002] Avian influenza (AI) is a highly contagious respiratory disease of poultry caused by influenza A virus of the Orthomyxoviridae family. The disease develops rapidly, spreads quickly, and has a wide range of prevalence, causing huge losses to my country's breeding industry. According to the different antigenic characteristics of hemagglutinin (HA) and neuraminidase (NA), avian influenza virus (AIV) is divided into many subtypes, including 18 HA subtypes (H1~H18) and 11 NA subtypes (N1~N11). Among them, H9N2 AIV is prevalent in various types of poultry around the world and can infect mammals and even humans. The cleavage site of H9N2 AIV HA protein has only one or two discontinuous basic amino acids. It is a low-pathogenic avian influenza virus. H9N2 AIV infection can only cause mild respiratory symptoms in chickens. However, H9N2 AIV infection can lead to secondary infection with pathogens such as Escherichia coli, Mycoplasma gallisepticum and infectious bronchitis virus, causing obvious respiratory symptoms or death in chickens.

[0003] Sick and infected birds are the main sources of avian influenza, followed by recovered birds. The virus can be transmitted through the secretions and excretions of the respiratory and digestive tracts of sick and infected birds. The cages, feed troughs, and water troughs of sick and infected birds can also be sources of infection. Insects and various wild birds carrying the virus can become AIV carriers. The most common way of AIV transmission is horizontal transmission. The main route of transmission is through the respiratory tract within susceptible animal groups. The feces of sick birds also carry the virus and can also contaminate normal birds. The main symptoms of poultry are changes in the respiratory tract, reproductive system, and digestive tract.

[0004] In practice, vaccination is the main method used to prevent avian influenza outbreaks. However, the avian influenza virus is highly variable, so there are many serotypes at present, which makes it difficult to develop vaccines. The existing inactivated vaccines have a long immune blank period and can only stimulate humoral immunity, not cellular immunity and mucosal immune response. Therefore, they cannot effectively inhibit the replication and spread of avian influenza virus in chickens. Studies have reported that after immunizing chickens with inactivated vaccines, even if the HI antibody titer in the body is as high as 2 10-12, animals still have the problem of virus transmission. In addition, the widespread vaccination used to control AIV epidemics in poultry favors the drift of seasonal vaccine strains to antigenically diverse viruses, which may reduce vaccine efficacy and lead to vaccination failure. In comparison, mucosal vaccines induce better local immune responses. Given that AIV infection and spread occur on the mucosal surface, administering vaccines via the mucosal route can induce mucosal immunity to provide the first line of defense against viral infection. The antibodies induced by mucosal immunity have a wider range of effects, and compared with serum IgG antibodies, sIgA antibodies have a stronger effect in protecting against infection with influenza virus variants.

[0005] In recent years, with the development of veterinary medicine and molecular biology technology, lactic acid bacteria have been widely used as mucosal surface antigen presentation carriers in the prevention and control of bacteria, viruses, parasites and other fields. Lactic acid bacteria are safe, endotoxin-free, and the exogenous proteins expressed do not need to be purified and can be taken orally together with the bacteria. They are easy to operate and cause less stress to animals, avoiding blood-borne transmission caused by intramuscular injections and reducing the risk of disease transmission. Compared with injectable vaccines, the increase in local mucosal immunity after oral immunization builds the first "firewall" for the body to deal with pathogens. At the same time, it has the potential to block the further spread and spread of pathogens.

[0006] Yang Wentao from Jilin Agricultural University constructed a recombinant lactic acid bacteria NC8-pSIP409-NP-M1-DCpep based on the genes of avian influenza virus NP, M1 and dendritic cell-inducing peptides, which can induce specific T cell immune responses and mucosal immune responses in mice, and can produce partial protection against the same type of mouse-adapted avian influenza virus and heterologous influenza virus. Shi Shaohua from Jilin Agricultural University used Lactobacillus plantarum as a live bacterial carrier for presenting antigens, the gene fragment of the HA protein, the main immune protective antigen of avian influenza virus, as the target gene, and fused a 36bp dendritic cell-inducing peptide (Dcpep) to construct a recombinant Lactobacillus plantarum expression system expressing HA-DC, which has good immunogenicity and can induce good mucosal immune responses and systemic immune responses.

[0007] The above research has laid the foundation for the development of oral recombinant lactic acid bacteria live vector vaccine for avian influenza virus. However, H9N2AIV infection can also induce immunosuppression in chickens, leading to vaccine immunization failure in chickens. Currently, there are few reports on relieving immunosuppression. Summary of the invention

[0008] In view of the above-mentioned prior art, the purpose of the present invention is to provide a recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein and its application. The present invention uses a combination of recombinant lactic acid bacteria H9-HA expressing H9N2 subtype avian influenza virus HA protein and recombinant lactic acid bacteria H9-NP-M1 expressing H9N2 subtype avian influenza virus NP protein and M1 protein, which can effectively alleviate the body's immunosuppression, improve the effect of traditional inactivated vaccines, reduce the viral load in the body after infection with H9N2 subtype avian influenza virus, and has good application prospects in the prevention and treatment of avian influenza.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention provides a recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein, comprising: recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1;

[0011] The recombinant lactic acid bacteria H9-HA expresses the HA protein of the H9N2 subtype avian influenza virus; and the recombinant lactic acid bacteria H9-NP-M1 expresses the NP protein and the M1 protein of the H9N2 subtype avian influenza virus.

[0012] In the recombinant lactic acid bacteria composition, the viable counts of the recombinant lactic acid bacteria H9-HA and the recombinant lactic acid bacteria H9-NP-M1 are both greater than or equal to 10 10 CFU / g.

[0013] Preferably, in the recombinant lactic acid bacteria composition, the ratio of the number of viable cells of the recombinant lactic acid bacteria H9-HA and the recombinant lactic acid bacteria H9-NP-M1 is 1:1.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned recombinant lactic acid bacteria composition, comprising the following steps:

[0015] (1) Preparation of recombinant lactic acid bacteria H9-HA fermentation broth:

[0016] The recombinant lactic acid bacteria H9-HA was inoculated into MRS liquid culture medium for fermentation culture at a temperature of 25-35° C., an inducer was added 4 hours after inoculation, and culture was continued for 6-10 hours to prepare a fermentation liquid of the recombinant lactic acid bacteria H9-HA;

[0017] (2) Preparation of recombinant lactic acid bacteria H9-NP-M1 fermentation broth:

[0018] The recombinant lactic acid bacteria H9-NP-M1 was inoculated into MRS liquid culture medium for fermentation culture at a temperature of 25-35° C., an inducer was added 4 hours after inoculation, and culture was continued for 6-10 hours to prepare a fermentation liquid of the recombinant lactic acid bacteria H9-NP-M1;

[0019] (3) Preparation of recombinant lactic acid bacteria composition:

[0020] The recombinant lactic acid bacteria H9-HA fermentation broth and the recombinant lactic acid bacteria H9-NP-M1 fermentation broth are centrifuged separately, the bacterial precipitates are collected, the bacterial precipitates are mixed with a lyophilization protective agent, and vacuum freeze-dried to obtain recombinant lactic acid bacteria H9-HA bacterial powder and recombinant lactic acid bacteria H9-NP-M1 bacterial powder, respectively; the recombinant lactic acid bacteria H9-HA bacterial powder and the recombinant lactic acid bacteria H9-NP-M1 bacterial powder are compounded to prepare a recombinant lactic acid bacteria composition.

[0021] Preferably, in step (1) and step (2), the inducer added is sake lactobacillus, and the final concentration of the inducer after addition is 100 ng / mL.

[0022] Preferably, in step (3), the lyophilization protective agent is composed of the following raw materials in parts by weight:

[0023] 22.5 parts of skimmed milk powder, 9 parts of sucrose, 1 part of glycerol, and 67.5 parts of water.

[0024] Preferably, in step (3), the bacterial precipitate is mixed with a lyophilization protective agent in a mass ratio of 1:1 and vacuum freeze-dried for 48 hours.

[0025] Preferably, in step (3), the number of viable bacteria in the recombinant lactic acid bacteria H9-HA powder and the recombinant lactic acid bacteria H9-NP-M1 powder is greater than or equal to 10 11 CFU / g.

[0026] Preferably, in step (3), according to the viable bacterial counts of the recombinant lactic acid bacteria H9-HA powder and the recombinant lactic acid bacteria H9-NP-M1 powder, the recombinant lactic acid bacteria H9-HA powder and the recombinant lactic acid bacteria H9-NP-M1 powder are respectively weighed and compounded, so that the viable bacterial counts of the recombinant lactic acid bacteria H9-HA and the recombinant lactic acid bacteria H9-NP-M1 in the compounded recombinant lactic acid bacteria composition are both 1×10 10 CFU / g.

[0027] The third aspect of the present invention provides the use of the above-mentioned recombinant lactic acid bacteria composition in at least one of the following (1)-(3):

[0028] (1) Preparation of microecological preparations that relieve immune suppression;

[0029] (2) Preparing a promoter for enhancing the effect of avian influenza virus vaccine;

[0030] (3) Prepare drugs to reduce the viral load in the body after infection with avian influenza virus.

[0031] In the above application, the body's immunosuppression is caused by H9N2 AIV infection or by the cyclophosphamide immunosuppressant.

[0032] In the above application, the avian influenza virus is an H9N2 subtype avian influenza virus.

[0033] The fourth aspect of the present invention provides an avian influenza immune microecological preparation, wherein the avian influenza immune microecological preparation uses the above-mentioned recombinant lactic acid bacteria composition as an active ingredient.

[0034] Furthermore, the avian influenza immune microecological preparation also includes auxiliary materials, and the auxiliary materials are selected from one or more of glucose, lactose, fructose, sodium alginate, and maltodextrin.

[0035] Furthermore, the avian influenza immune microecological preparation can be prepared into different dosage forms according to the requirements of use and storage, such as liquid preparations, powders, granules, capsules, etc.

[0036] Beneficial effects of the present invention:

[0037] (1) In order to solve the problem that H9N2 AIV infection induces immunosuppression in chickens and causes vaccine immunization failure in chickens, the present invention combines recombinant lactic acid bacteria H9-HA expressing H9N2 subtype avian influenza virus HA protein with recombinant lactic acid bacteria H9-NP-M1 expressing H9N2 subtype avian influenza virus NP protein and M1 protein. The combination can significantly alleviate the body's immunosuppression and has a synergistic effect in reducing the increase in mortality caused by immunosuppression compared with a single recombinant lactic acid bacteria. Moreover, the combination of recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1 can synergistically improve the level of H9 subtype avian influenza antibodies and mucosal antibody sIgA.

[0038] (2) When used together with traditional inactivated vaccines, the recombinant lactic acid bacteria composition of the present invention can also improve the immune effect of traditional inactivated avian influenza vaccines.

[0039] (3) The recombinant lactic acid bacteria composition of the present invention can also reduce the viral load in the body after infection with the H9N2 subtype avian influenza virus, and has good application prospects in the prevention and treatment of the H9N2 subtype avian influenza virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Secretory immunoglobulin A (sIgA) content in intestinal mucosa in Experimental Example 1; * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001;

[0041] Figure 2: sIgA content in intestinal mucosa in Experiment 3; * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001;

[0042] Figure 3 : Fluorescence quantitative RT-PCR standard curve;

[0043] Figure 4 :Fluorescence quantitative PCR detection of viral copy numbers in cloacal cotton swabs of each group; * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001; DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments. If the specific experimental conditions are not specified in the embodiments, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained through commercial channels. Among them:

[0046] The recombinant lactic acid bacteria H9-HA expressing the HA protein of the H9N2 subtype avian influenza virus is constructed with reference to the method described in the first chapter of the second part of "Study on the protective effect of recombinant HA-DC lactic acid bacteria mediated by dendritic cells against H9N2 avian influenza" (Master's thesis of Jilin Agricultural University, Shi Shaohua, 2013) on the construction of recombinant lactic acid bacteria expressing HA-DC. The recombinant lactic acid bacteria H9-HA in the present invention corresponds to the recombinant lactic acid bacteria expressing HA-DC constructed in the master's thesis.

[0047] The recombinant lactic acid bacteria H9-NP-M1 expressing the NP protein and M1 protein of the H9N2 subtype avian influenza virus was constructed with reference to the method described in the first chapter of the second part of "Research on recombinant lactic acid bacteria targeting universal avian influenza virus of dendritic cells" (doctoral dissertation of Jilin Agricultural University, Yang Wentao, 2015) on the construction and verification of recombinant lactic acid bacteria expressing NP-M1-Dcpep. The recombinant lactic acid bacteria H9-NP-M1 in the present invention corresponds to the recombinant lactic acid bacteria NC8-pSIP409-NP-M1-DCpep constructed in the doctoral dissertation.

[0048] Example 1: Study on the fermentation process of recombinant lactic acid bacteria expressing H9N2 subtype avian influenza virus antigen protein

[0049] 1. Test materials

[0050] Strains: recombinant lactic acid bacteria H9-HA, recombinant lactic acid bacteria H9-NP-M1.

[0051] Culture medium:

[0052] MRS liquid culture medium: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, Tween-80 1 mL / L, pH 6.0±0.05, sterilization at 121°C for 20 min.

[0053] Sake lactobacillus was purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0054] 2. Test methods

[0055] (1) Preparation of primary seed solution: 0.5 mL of the glycerol tube storage solution of recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1 was inoculated into 100 mL of MRS liquid culture medium, cultured at 30°C overnight, and then transferred to 100 mL of freshly sterilized MRS liquid culture medium at a 3% (volume ratio) inoculation amount, and cultured at 30°C overnight to prepare primary seed solutions.

[0056] (2) Preparation of secondary seed solution: The primary seed solution was inoculated into 3LMRS liquid culture medium at a volume ratio of 3% at 30° C. for overnight culture to obtain the secondary seed solution.

[0057] (3) Seed tank activation: 3 L of secondary seed liquid was inoculated into a 100 L seed tank (containing 60 L of MRS liquid medium), and activated at 30 °C for 10-12 h under anaerobic conditions to obtain a seed tank fermentation liquid;

[0058] (4) Large tank fermentation process: 60L of the activated seed tank fermentation liquid is transferred to a 500L fermentation tank (containing 400L of MRS liquid culture medium), cultured at 30°C for 4h under anaerobic conditions, and then added with sake lactobacillus for induction culture. The final concentration of sake lactobacillus after addition is 100ng / mL. The fermentation ends when the pH of the fermentation liquid drops below 4.0 (or the pH value does not continue to decrease). The total fermentation time is generally controlled at about 10-12h. Samples are taken every 2h during the fermentation process to detect and record the OD value of the fermentation liquid. 600 , pH value and viable bacterial count.

[0059] 3. Test results

[0060] (1) Fermentation effect of recombinant lactic acid bacteria H9-HA

[0061] The fermentation data of recombinant lactic acid bacteria H9-HA are shown in Table 1.

[0062] Table 1 Fermentation data of recombinant lactic acid bacteria H9-HA

[0063]

[0064] As can be seen from Table 1, the number of viable cells of recombinant lactic acid bacteria H9-HA in the stable period was as high as 5.2×10 9 CFU / mL.

[0065] (2) Fermentation effect of recombinant lactic acid bacteria H9-NP-M1

[0066] The fermentation data of the recombinant lactic acid bacteria H9-NP-M1 are shown in Table 2.

[0067] Table 2 Fermentation data of recombinant lactic acid bacteria H9-NP-M1

[0068]

[0069]

[0070] As can be seen from Table 2, the number of viable bacteria in the stable period of the recombinant lactic acid bacteria H9-NP-M1 fermentation process reached a maximum of 7.1×10 9 CFU / mL.

[0071] Example 2: Preparation of a recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein

[0072] (1) Preparation of recombinant lactic acid bacteria H9-HA fermentation broth:

[0073] The recombinant lactic acid bacteria H9-HA was sequentially subjected to primary seed liquid preparation, secondary seed liquid preparation and seed tank activation, and the operation was the same as in Example 1; the activated recombinant lactic acid bacteria H9-HA seed liquid was inoculated into MRS liquid culture medium for fermentation culture, the inoculation amount was 15% of the volume of the MRS liquid culture medium, the fermentation culture temperature was 30°C, and the inducer sake lactobacillus was added 4 hours after inoculation, the final concentration of the inducer after addition was 100 ng / mL, and the culture was continued for 6 hours to prepare the recombinant lactic acid bacteria H9-HA fermentation liquid.

[0074] (2) Preparation of recombinant lactic acid bacteria H9-NP-M1 fermentation broth:

[0075] The recombinant lactic acid bacteria H9-NP-M1 was sequentially subjected to primary seed liquid preparation, secondary seed liquid preparation and seed tank activation, and the operation was the same as in Example 1; the activated recombinant lactic acid bacteria H9-NP-M1 seed liquid was inoculated into MRS liquid culture medium for fermentation culture, the inoculation amount was 15% of the volume of the MRS liquid culture medium, the fermentation culture temperature was 30°C, and the inducer sake lactobacillus was added 4 hours after inoculation, the final concentration of the inducer after addition was 100 ng / mL, and the culture was continued for 6 hours to prepare the recombinant lactic acid bacteria H9-NP-M1 fermentation liquid.

[0076] (3) Preparation of recombinant lactic acid bacteria H9-HA powder:

[0077] The fermentation liquid of the recombinant lactic acid bacteria H9-HA was centrifuged to collect the bacterial precipitate, and the bacterial precipitate was mixed with a lyophilization protective agent at a mass ratio of 1:1, wherein the lyophilization protective agent was composed of the following raw materials in parts by mass:

[0078] 22.5 parts of skimmed milk powder, 9 parts of sucrose, 1 part of glycerol, and 67.5 parts of water.

[0079] Then, the recombinant lactic acid bacteria H9-HA powder was obtained by freeze-drying for 48 hours using conventional vacuum freeze-drying technology; the number of viable bacteria in the recombinant lactic acid bacteria H9-HA powder was not less than 1×10 11 CFU / g.

[0080] (4) Preparation of recombinant lactic acid bacteria H9-NP-M1 powder:

[0081] The fermentation liquid of the recombinant lactic acid bacteria H9-NP-M1 was centrifuged, the bacterial precipitate was collected, and the bacterial precipitate was mixed with a lyophilization protective agent at a mass ratio of 1:1, wherein the lyophilization protective agent was composed of the following raw materials in parts by mass:

[0082] 22.5 parts of skimmed milk powder, 9 parts of sucrose, 1 part of glycerol, and 67.5 parts of water.

[0083] Then, the recombinant lactic acid bacteria H9-NP-M1 powder was obtained by freeze-drying for 48 hours using conventional vacuum freeze-drying technology; the number of viable bacteria in the recombinant lactic acid bacteria H9-NP-M1 powder was not less than 1×10 11 CFU / g.

[0084] (5) Preparation of recombinant lactic acid bacteria composition:

[0085] According to the viable bacterial counts of the recombinant lactic acid bacteria H9-HA powder and the recombinant lactic acid bacteria H9-NP-M1 powder, appropriate amounts of the powders were weighed and mixed respectively, so that the viable bacterial counts of the recombinant lactic acid bacteria H9-HA and the recombinant lactic acid bacteria H9-NP-M1 in the mixed powders were both 1×10 10 CFU / g.

[0086] Experimental Example 1: Establishment of cyclophosphamide immunosuppression model and investigation of the immunosuppression effect of the body

[0087] 1. Test materials

[0088] Cyclophosphamide immunosuppressant: purchased from Shanghai Yuanye Biotechnology Co., Ltd., model S30563-5G.

[0089] Recombinant Newcastle disease virus and avian influenza virus (H9 subtype) bivalent inactivated vaccine (referred to as "Newcastle disease bivalent inactivated vaccine") was purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0090] Experimental animals: 1-day-old AA white-feather broiler chickens, fed with broiler feed 510 before 20 days of age, and fed with broiler feed 511 after 21 days of age. Broiler feed 510 and broiler feed 511 were both purchased from Shannong Agriculture and Animal Husbandry (Tai'an) Co., Ltd. and do not contain antibiotics.

[0091] 2. Test methods

[0092] 320 one-day-old chicks were randomly divided into 4 groups: normal control group, inactivated vaccine group, low-dose immunosuppression group, and high-dose immunosuppression group; each group had 2 parallels, each with 40 chicks, parallel 1 was used for clinical symptom (mortality), HI antibody and cytokine determination; parallel 2 was used for determination of immune organ index and sIgA. The treatment of each group was specifically based on Table 3.

[0093] Table 3 Experimental grouping and design

[0094]

[0095] Monitoring indicators:

[0096] (1) Clinical symptoms: Clinical symptoms and animal deaths were observed and recorded daily;

[0097] (2) HI antibody: At 7, 14, 21, 28, 35 and 42 days of age, 10 chickens were randomly selected from each group in parallel, 1 mL of blood was collected from the subwing vein, and serum was separated at 4500 rpm for 10 min for the determination of hemagglutination inhibition (HI) antibodies.

[0098] (3) Cytokine determination: At 7, 14, 21, 28, 35, and 42 days of age, 10 chickens were randomly selected from each group in parallel, 1 mL of blood was collected from the subwing vein, and serum was separated at 4500 rpm for 10 min. The chicken interleukin 10 (IL-10) ELISA detection kit was used to determine the IL-10 level in serum;

[0099] (4) Immune organ index determination: At 14, 28, and 42 days of age, four chickens were randomly selected from each group and euthanized. The spleen and bursa of Fabricius were weighed and the immune organ index was calculated.

[0100] (5) Determination of secretory immunoglobulin A (sIgA): At 14, 28 and 42 days of age, 4 chickens were randomly selected from each group for euthanasia (the selected samples were the same as those for the determination of immune organ index), and the jejunal intestinal segments were selected to obtain intestinal mucosa. The level of intestinal mucosal sIgA was detected using the chicken secretory immunoglobulin sIgA ELISA detection kit. (Method: Remove a jejunal intestinal segment of about 20 cm in length, rinse it with 60 mL of 0.9% saline for 4 times, cut the intestinal segment under sterile conditions, gently absorb the remaining chyme and fecal fluid with absorbent paper, and gently scrape the intestinal mucus with a blade into a 10 mL centrifuge tube, add 2 times the volume of 0.9% saline, shake and mix thoroughly at low temperature for 30 minutes, and centrifuge at 12000 rpm and 4°C for 20 minutes. All the supernatant obtained was frozen at -20°C for later use.

[0101] 3. Test results

[0102] (1) Clinical symptoms

[0103] By observing the clinical symptoms, it was found that after the injection of cyclophosphamide, both the low-dose immunosuppression group and the high-dose immunosuppression group showed symptoms such as slow growth of chickens, reduced excitability of chicks, decreased appetite, weight loss, ruffled feathers, listlessness, individual weakness, and death.

[0104] (2) Mortality

[0105] The statistical results of mortality are shown in Table 4. The mortality rate in the high-dose immunosuppression group was higher than that in the low-dose immunosuppression group, and both were significantly higher than those in the inactivated vaccine group and the normal control group.

[0106] Table 4 Mortality statistics

[0107]

[0108] (3) HI antibody

[0109] The HI antibody monitoring results are shown in Table 5.

[0110] Table 5 H9 subtype avian influenza HI antibody levels (Log2) in different age groups

[0111]

[0112]

[0113] Note: ** represents P value < 0.01; *** represents P value < 0.001;

[0114] As shown in Table 5, as the maternal antibodies in the body continued to decrease, the antibodies in the normal control group showed a continuous downward trend, and were significantly lower than those in the inactivated vaccine group at 28-42 days of age; after the inactivated vaccine group was injected with inactivated vaccine at 7 days of age, the antibodies began to show a gradual upward trend after 21 days of age; compared with the inactivated vaccine group, the immunosuppressive group was significantly lower than the inactivated vaccine group after 21 days of age due to the injection of cyclophosphamide inhibitors, and the antibody level in the high-dose immunosuppressive group was lower than that in the low-dose immunosuppressive group.

[0115] (4) Immune organ index

[0116] Immune organ index = weight of each immune organ (g) / live body weight (g) × 100. The immune organ monitoring results are shown in Table 6-7.

[0117] Table 6 Spleen Index

[0118]

[0119] Note: * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001

[0120] As can be seen from Table 6: compared with the inactivated vaccine group, cyclophosphamide significantly reduced the spleen index of chickens at 14 and 28 days of age, and the inhibitory effect was positively correlated with the cyclophosphamide dose. At 42 days of age, the inhibitory effect weakened, and only the spleen index of the high-dose immunosuppression group and the inactivated vaccine group showed a statistical difference.

[0121] Table 7 Bursal index

[0122]

[0123] Note: ** represents P value < 0.01; *** represents P value < 0.001

[0124] It can be seen from Table 7 that compared with the inactivated vaccine group, cyclophosphamide can significantly reduce the bursal index at 14 and 28 days of age, and the immunosuppressive effect is weakened at 42 days of age. Consistent with literature reports, cyclophosphamide can thin the cortex of the bursal follicle of chickens, reduce the number of lymphocytes in the cortex, make the epithelial boundaries unclear, and necrotic vacuoles can be seen in the nodules and mucosal layers. Cyclophosphamide has a serious inhibitory effect on mitotic mother cells, and the bursa of Fabricius is the central immune organ of chickens and the place where B cells differentiate and mature, so cyclophosphamide has a strong damaging effect on the bursa of Fabricius.

[0125] (5) Serum IL-10 level

[0126] The results of serum IL-10 monitoring are shown in Table 8.

[0127] Table 8 Effect of cyclophosphamide on serum IL-10 (ng / L)

[0128]

[0129] Note: * represents P value < 0.05, *** represents P value < 0.001

[0130] As shown in Table 8, at 14 days of age, the IL-10 content in the serum of the high-dose immunosuppression group was significantly lower than that of the inactivated vaccine group; at 21 days and 28 days of age, the IL-10 content of the inactivated vaccine group was significantly increased compared with the normal control group, but the IL-10 content of the low-dose immunosuppression group and the high-dose immunosuppression group was significantly decreased compared with the inactivated vaccine group, indicating that the use of cyclophosphamide immunosuppressant inhibited the body's immune response caused by immunization with inactivated vaccine, and the effect of the high-dose immunosuppression group was more obvious.

[0131] (6) Secretory immunoglobulin A (sIgA) level

[0132] The results of monitoring sIgA in jejunal intestinal mucosa are shown in Tables 9 and Figure 1 .

[0133] Table 9 Secretory immunoglobulin A (sIgA) level (ng / mL)

[0134]

[0135] Note: * represents P value < 0.05; *** represents P value < 0.001

[0136] From Table 9 and Figure 1 The results showed that at 14 days of age, there was a statistically significant difference in the sIgA level between the high-dose immunosuppression group and the inactivated vaccine group. At 28 and 42 days of age, the sIgA levels in the low-dose and high-dose groups were significantly lower than those in the inactivated vaccine group, indicating that after cyclophosphamide inhibited the body's immune function, the sIgA level in the jejunal mucosa was also significantly reduced.

[0137] In summary, the results showed that injection of cyclophosphamide would inhibit the growth of experimental animals and cause death, affect the growth and development of immune organs, inhibit the body's immune response, and inhibit the production of IL-10 and secretory immunoglobulin A (sIgA) in the mucosa after immunization with inactivated vaccine, indicating that the cyclophosphamide immunosuppression model was successfully established, and a low dose (80 mg / kg chicken weight) could cause obvious immunosuppression, so 80 mg / kg was selected for subsequent experiments.

[0138] Test Example 2: Test on the effect of recombinant lactic acid bacteria composition on relieving immune suppression

[0139] 1. Test materials

[0140] drug:

[0141] Recombinant lactic acid bacteria H9-HA powder: The recombinant lactic acid bacteria H9-HA powder prepared in Example 2 of the present invention was diluted with carrier glucose to a viable count of 1×10 10 CFU / g, and the recombinant lactic acid bacteria H9-HA powder was obtained, which was prepared into a solution with sterile water and administered orally according to the dosage in Table 10.

[0142] Recombinant lactic acid bacteria H9-NP-M1 powder: The recombinant lactic acid bacteria H9-NP-M1 powder prepared in Example 2 of the present invention was diluted with carrier glucose to a viable count of 1×10 10 CFU / g, and the recombinant lactic acid bacteria H9-NP-M1 powder was obtained, which was prepared into a solution with sterile water and administered orally according to the dosage in Table 10.

[0143] Recombinant lactic acid bacteria composition: The recombinant lactic acid bacteria composition prepared in Example 2 of the present invention was prepared into a solution with sterile water and administered intragastrically according to the dosage in Table 10.

[0144] The method for determining the number of viable bacteria in the recombinant lactic acid bacteria composition, recombinant lactic acid bacteria H9-HA powder and recombinant lactic acid bacteria H9-NP-M1 powder prepared in Example 2 is as follows: weigh 1 g of the powder, add 99 mL of sterile saline and shake for 30 minutes, then take 1 mL of the dilution gradient to count the number of viable bacteria.

[0145] Cyclophosphamide immunosuppressant: purchased from Shanghai Yuanye Biotechnology Co., Ltd., model S30563-5G.

[0146] Inactivated vaccine: Recombinant Newcastle disease virus and avian influenza virus (H9 subtype) bivalent inactivated vaccine (referred to as “Newcastle disease bivalent inactivated vaccine”) was purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0147] Experimental animals: 1-day-old AA white-feather broiler chickens, fed with broiler feed 510 before 20 days of age, and fed with broiler feed 511 after 21 days of age, all of which were purchased from Shannong Agriculture and Animal Husbandry (Tai'an) Co., Ltd. and did not contain antibiotics.

[0148] 2. Test methods

[0149] 540 one-day-old chicks were randomly divided into 9 groups, with 2 parallels in each group and 30 chicks in each parallel. Parallel 1 was used for mortality statistics, HI antibody and cytokine monitoring; parallel 2 was used to determine the sIgA content in the intestinal mucosa. The experimental design is as follows:

[0150] Table 10 Experimental groups and treatments

[0151]

[0152]

[0153] Note: Immunization dose of inactivated vaccine: subcutaneous injection of 0.2 mL / animal according to the instructions.

[0154] The mortality rate, HI antibody level, secretory immunoglobulin A (sIgA) level and serum IL-10 level of each treatment group were counted and monitored according to the method of Experimental Example 1.

[0155] 3. Test results

[0156] (1) Mortality

[0157] The mortality statistics of each treatment group are shown in Table 11.

[0158] Table 11 Statistical results of mortality in each group

[0159]

[0160] As shown in Table 11, chickens died in the groups injected with the cyclophosphamide immunosuppressant. Comparative analysis of the groups showed that the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition was better than any one of the recombinant lactic acid bacteria alone in alleviating the mortality caused by immunosuppression, and had a synergistic effect.

[0161] (2) HI antibody level

[0162] The HI antibody levels of each treatment group are shown in Table 12.

[0163] Table 12 H9 subtype avian influenza HI antibody levels (Log 2) in different groups at different ages

[0164]

[0165]

[0166] Note: *** represents P value < 0.001

[0167] As can be seen from Table 12, after inoculation with inactivated vaccine at 7 days of age, the antibody levels of H9 subtype avian influenza in groups 2, 3, 4 and 5 all showed an upward trend after 21 days of age, and the antibody level in group 3 was the highest. However, due to the injection of cyclophosphamide, the antibody levels of groups 6, 7, 8 and 9 were all lower than those in the inactivated vaccine group, indicating that cyclophosphamide inhibited the body's immune function. Compared with group 6, the antibody levels of groups 7, 8 and 9 were improved, among which the improvement effect of group 7 was better than that of groups 8 and 9, and was significantly higher than that of group 6 (28 days old), indicating that the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition is better than any of the recombinant lactic acid bacteria alone in alleviating immunosuppression and improving the vaccine antibody level.

[0168] (3) Secretory immunoglobulin A (sIgA) level

[0169] The levels of mucosal antibody sIgA in each treatment group are shown in Table 13.

[0170] Table 13 Secretory immunoglobulin A (sIgA) content (ng / mL)

[0171]

[0172] Note: * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001

[0173] As shown in Table 13, at 21 and 28 days of age, the sIgA content in Group 3 was significantly increased compared with Group 2, while the sIgA level in Groups 4 and 5 was significantly increased only at 28 days of age, indicating that the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition can significantly improve the mucosal immune level, and the effect is better than using any of the recombinant lactic acid bacteria alone. Compared with Group 6, the sIgA level in Group 7 was significantly increased at 21, 28, and 35 days of age, indicating that under immunosuppression, the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition can significantly improve the mucosal immune level.

[0174] (4) Serum IL-10 level

[0175] The serum IL-10 levels of each treatment group are shown in Table 14.

[0176] Table 14 Serum IL-10 levels in each group (ng / L)

[0177]

[0178]

[0179] Note: * represents P value < 0.05; ** represents P value < 0.01; *** represents P value < 0.001

[0180] As shown in Table 14, at 14, 21 and 28 days of age, the IL-10 content in Group 3 was significantly higher than that in Group 2, and the effect of increasing the IL-10 content was better than that in Groups 4 and 5, indicating that the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition can significantly increase the level of cytokine IL-10. At 21 and 28 days of age, the IL-10 level in Group 7 was significantly increased compared with Group 6, while the values ​​of Groups 8 and 9 were increased but not significantly different compared with Group 6, indicating that under the condition of immunosuppression, the (H9-HA) + (H9-NP-M1) recombinant lactic acid bacteria composition can promote the secretion of IL-10.

[0181] Experimental Example 3: Effect of recombinant lactic acid bacteria composition on the immune effect of traditional inactivated vaccines

[0182] 1. Test materials

[0183] Drug: The recombinant lactic acid bacteria composition prepared in Example 2 of the present invention was prepared into a solution with sterile water and administered intragastrically according to the dosage in Table 15.

[0184] Inactivated vaccine: Recombinant Newcastle disease virus and avian influenza virus (H9 subtype) bivalent inactivated vaccine (referred to as “Newcastle disease bivalent inactivated vaccine”) was purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0185] Experimental animals: 1-day-old AA white-feather broiler chickens, fed with broiler feed 510 before 20 days of age, and fed with broiler feed 511 after 21 days of age, all of which were purchased from Shannong Agriculture and Animal Husbandry (Tai'an) Co., Ltd. and did not contain antibiotics.

[0186] 2. Test methods

[0187] 180 experimental animals were randomly divided into 6 groups, 30 in each group, including a normal control group, an inactivated vaccine group, an experimental group 1, an experimental group 2, an experimental group 3, and an experimental group 4. Each group was treated according to Table 15. The dosage of the recombinant lactic acid bacteria composition was 500 million CFU / animal / day, and the inactivated vaccine was subcutaneously injected at 0.2 mL / animal.

[0188] Table 15 Experimental groups and treatments

[0189]

[0190]

[0191] The HI antibody level and mucosal antibody sIgA level of each test group at different times were statistically analyzed and monitored according to the method of Test Example 1.

[0192] 3. Test results

[0193] (1) Antibodies to H9 subtype avian influenza

[0194] The H9 subtype avian influenza antibody levels in each treatment group at different time points were detected by hemagglutination inhibition test. The results are shown in Table 16.

[0195] Table 16 H9 subtype avian influenza HI antibody levels (Log2) in different groups at different ages

[0196]

[0197] As shown in Table 16, after 21 days of age, the titers of test group 1 and test group 3 were higher than those of the inactivated vaccine group, and test group 1 was slightly higher than that of test group 3. The titer of test group 1 was up to 1.5 (at 42 days of age) higher than that of the inactivated vaccine group; after 21 days of age, the antibody levels of test groups 2 and 4 were slightly better than those of the normal control group. This indicates that the combined use of the recombinant lactic acid bacteria composition and the inactivated vaccine, or the use of the recombinant lactic acid bacteria composition alone, can increase the level of H9 subtype avian influenza antibodies.

[0198] (2) Secretory immunoglobulin A (sIgA) level

[0199] The results of the determination of mucosal antibody sIgA levels are shown in Tables 17 and Figure 2 .

[0200] Table 17 Content of intestinal mucosal secretory immunoglobulin A (sIgA) in each group at different ages (ng / mL)

[0201]

[0202] Note: ** represents P value < 0.01; *** represents P value < 0.001

[0203] From Table 17 and Figure 2 It can be seen that at 18 days and 25 days of age, the content of sIgA in the intestinal mucosa of test group 1 and test group 3 was significantly increased compared with the inactivated vaccine group (p < 0.001). At 18 days of age, compared with the inactivated vaccine group, the sIgA content of test group 1 increased by 24.4%, and the sIgA content of test group 3 increased by 24.0%; at 25 days of age, the sIgA content of test group 1 and test group 3 increased by 28.6% and 25.8% respectively compared with the inactivated vaccine group; similarly, at 18 days and 25 days of age, the content of sIgA in the intestinal mucosa of test group 2 and test group 4 was significantly increased compared with the normal control group. The above results show that the use of the recombinant lactic acid bacteria composition promotes mucosal immune response and increases the secretion of sIgA.

[0204] Experimental Example 4: Effect of the recombinant lactic acid bacteria composition on the viral load in the body after infection with H9N2 subtype avian influenza virus

[0205] 1. Test materials

[0206] Drug: the recombinant lactic acid bacteria composition prepared in Example 2 of the present invention; the recombinant lactic acid bacteria composition prepared in Example 2 was prepared into a solution with sterile water, and the solution was gavage at a dose of 250 million CFU / mouse / day for H9-HA and H9-NP-M1, respectively.

[0207] Inactivated vaccine: Recombinant Newcastle disease virus and avian influenza virus (H9 subtype) bivalent inactivated vaccine (referred to as “Newcastle disease bivalent inactivated vaccine”) was purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0208] Experimental animals: 1-day-old AA white-feather broiler chickens, fed with broiler feed 510 before 20 days of age, and fed with broiler feed 511 after 21 days of age, all of which were purchased from Shannong Agriculture and Animal Husbandry (Tai'an) Co., Ltd. and did not contain antibiotics.

[0209] 2. Test methods

[0210] 175 broilers were randomly divided into 7 groups, 25 in each group. At 18 days of age, except for the normal control group, the other groups were intranasally injected with H9N2 subtype avian influenza virus (EID50 = 10 -7 / 0.2mL) and treated with intramuscular injection of E. coli, with a live count of 10 6 CFU / mL, the challenge dose for all groups was 1 mL of each pathogenic microorganism, and the experimental grouping and treatment are shown in Table 18. The chickens were observed for 2 weeks after the challenge, during which the number of deaths was recorded and the mortality rate was calculated. Five chickens were randomly selected from each group 2, 4, and 6 days after the challenge, and the virus was taken by cloacal cotton swab for quantitative detection of viral load.

[0211] Table 18 Experimental groups and treatments

[0212]

[0213]

[0214] 3. Test results

[0215] (1) Mortality

[0216] The mortality statistics of each group are shown in Table 19.

[0217] Table 19 Statistical results of mortality in each group (within two weeks of infection)

[0218]

[0219] As shown in Table 19, the mortality rate of the challenge group was 40%, and the mortality rate of the inactivated vaccine group was 28%, which was 12% lower than that of the challenge control group. The mortality rates of the test groups 1 and 3 were 20% and 24%, respectively, which were 20% and 16% lower than those of the challenge control group; and the mortality rates of the test groups 2 and 4 were 8% and 4% lower than those of the challenge control group, respectively. This indicates that the recombinant lactic acid bacteria composition, whether used alone or in combination with an inactivated vaccine, can reduce the mortality caused by avian influenza virus infection.

[0220] (2) H9N2 subtype avian influenza virus load in cloacal cotton swabs

[0221] Fluorescence quantitative PCR was used to quantitatively determine the H9N2 subtype avian influenza virus load in cloacal cotton swabs. First, a fluorescence quantitative RT-PCR standard curve was established. The standard curve equation was Y = -3.682X + 46.396, with a good linear relationship and a correlation coefficient (R2) of 0.999 ( Figure 3 ). The correlation coefficient r2 and slope K value of the standard curve meet the theoretical requirements. The copy number of the recombinant plasmid has a good linear relationship with the CT value, which can be used as the basis for absolute fluorescence quantification. The melting curve has a single peak, no primer dimers and non-specific amplification products, proving that the primers can specifically amplify the target gene. The amplification curve conforms to the standard "S" shape, with a clear inflection point, good overall curve parallelism, a flat baseline and no obvious upward trend.

[0222] The results of the fluorescence quantitative PCR detection of the virus copy number in the cloacal cotton swabs of each group are shown in Table 20 and Figure 4 .

[0223] Table 20 H9N2 subtype avian influenza virus load (copy number) in cloacal cotton swabs of each group

[0224]

[0225]

[0226] Note: * represents P value < 0.05; ** represents P value < 0.01

[0227] As can be seen from Table 20, compared with the challenge control group, the inactivated vaccine group had significantly reduced avian influenza gene copy numbers 2 and 4 days after challenge, while compared with the inactivated vaccine group, the number of virus copies in cotton swabs in Test Groups 1 and 3 were significantly reduced 2, 4, and 6 days after challenge; compared with the challenge group, the number of virus copies in cotton swabs in Test Groups 2 and 4 showed a downward trend, indicating that the recombinant lactic acid bacteria composition of the present invention can reduce the intestinal avian influenza virus load, and the effect is better when used in combination with a vaccine.

[0228] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a recombinant lactic acid bacteria composition expressing H9N2 subtype avian influenza virus antigen protein in the preparation of a probiotic preparation for alleviating cyclophosphamide-induced immunosuppression, characterized in that: The recombinant lactic acid bacteria composition comprises: recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1; The recombinant lactic acid bacteria H9-HA expresses the HA protein of the H9N2 subtype avian influenza virus; the recombinant lactic acid bacteria H9-NP-M1 expresses the NP protein and M1 protein of the H9N2 subtype avian influenza virus; In the recombinant lactic acid bacteria composition, the viable counts of the recombinant lactic acid bacteria H9-HA and the recombinant lactic acid bacteria H9-NP-M1 are both greater than or equal to 10 10 CFU / g; The ratio of the number of live bacteria of recombinant lactic acid bacteria H9-HA and recombinant lactic acid bacteria H9-NP-M1 was 1:

1.

2. The use according to claim 1, characterized in that: The recombinant lactic acid bacteria composition is prepared by the following method: (1) Preparation of recombinant lactic acid bacteria H9-HA fermentation broth: The recombinant lactic acid bacteria H9-HA was inoculated into MRS liquid culture medium for fermentation culture at a temperature of 25-35° C., an inducer was added 4 hours after inoculation, and culture was continued for 6-10 hours to prepare a fermentation liquid of the recombinant lactic acid bacteria H9-HA; (2) Preparation of recombinant lactic acid bacteria H9-NP-M1 fermentation broth: The recombinant lactic acid bacteria H9-NP-M1 was inoculated into MRS liquid culture medium for fermentation culture at a temperature of 25-35° C., an inducer was added 4 hours after inoculation, and culture was continued for 6-10 hours to prepare a fermentation liquid of the recombinant lactic acid bacteria H9-NP-M1; (3) Preparation of recombinant lactic acid bacteria composition: The recombinant lactic acid bacteria H9-HA fermentation broth and the recombinant lactic acid bacteria H9-NP-M1 fermentation broth are centrifuged separately, the bacterial precipitates are collected, the bacterial precipitates are mixed with a lyophilization protective agent, and vacuum freeze-dried to obtain recombinant lactic acid bacteria H9-HA bacterial powder and recombinant lactic acid bacteria H9-NP-M1 bacterial powder, respectively; the recombinant lactic acid bacteria H9-HA bacterial powder and the recombinant lactic acid bacteria H9-NP-M1 bacterial powder are compounded to prepare a recombinant lactic acid bacteria composition.

3. The use according to claim 2, characterized in that: In step (1) and step (2), the inducer added is sake lactobacillus, and the final concentration of the inducer after addition is 100 ng / mL.

4. The use according to claim 2, characterized in that: In step (3), the lyophilization protective agent is composed of the following raw materials in parts by weight: 22.5 parts of skimmed milk powder, 9 parts of sucrose, 1 part of glycerol, and 67.5 parts of water.

5. The use according to claim 2, characterized in that: In step (3), the number of viable bacteria in the recombinant lactic acid bacteria H9-HA powder and the recombinant lactic acid bacteria H9-NP-M1 powder is greater than or equal to 10 11 CFU / g.

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