CN112370524A

CN112370524A - Antiviral composition and preparation method and application thereof

Info

Publication number: CN112370524A
Application number: CN202011436669.7A
Authority: CN
Inventors: 刘延亭; 张美美; 宫瑞雪; 代发文; 赵宝凯
Original assignee: Cangzhou Weiji Animal Husbandry Co ltd; BEIJING VICA BIOTECHNOLOGY CO LTD
Current assignee: Cangzhou Weiji Animal Husbandry Co ltd; BEIJING VICA BIOTECHNOLOGY CO LTD
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-02-19

Abstract

The invention belongs to the technical field of new biological veterinary drugs, and particularly relates to an antiviral composition and a preparation method and application thereof. The composition mainly takes porcine alpha, lambda 3 interferon and CD40L as active units, and arginine, glycerol and the like as protective agents, wherein the porcine interferon alpha, lambda 3 and CD40L are expressed proteins obtained by utilizing a genetic engineering recombination technology, and are mixed with the protective agents according to the following final concentration ratio after affinity chromatography purification: 50-500 million units/ml of porcine alpha interferon, 0.5-5 million units/ml of porcine lambda 3 interferon, 10-40 ug/ml of CD40L 10, 0.02-0.1 mol/L of arginine, 1-5% of glycerol, 0.05-0.2 mol/L of mannitol and 1-3% of trehalose. The composition has very good antiviral activity, can obviously inhibit the proliferation of porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus and the like, can be used as an immunomodulator, can obviously improve the humoral and cellular immunity level after immunization, and has long storage life and convenient use.

Description

Antiviral composition and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an antiviral composition and a preparation method and application thereof.

Background

With the development of large-scale breeding, viral diseases become the largest factor harming breeding industry, and the existing animal vaccines cannot completely prevent and control the diseases and lack effective treatment means. In recent years, various outbreaks of infectious diseases cause great harm to the pig industry in China, and some diseases which cause serious threats to pigs, such as swine fever, foot-and-mouth disease, reproductive and respiratory syndrome and the like, are not comprehensively controlled. Especially, outbreak of swine fever in China in 2018 makes the pig industry heavily lost.

The medicines for preventing and treating animal epidemic diseases in the market at present mainly comprise preventive vaccines and therapeutic antiviral chemical medicines. The safe and effective vaccine plays an important role in preventing major animal epidemic diseases, such as prevention and control of swine fever, foot-and-mouth disease, avian influenza and other epidemic diseases, and has a definite and reliable effect, but the vaccine has some defects, such as that an animal body only generates response aiming at a specific antigen, and various vaccines are required to be continuously immunized for various epidemic diseases; some vaccines have insufficient safety and effectiveness, such as highly pathogenic porcine reproductive and respiratory syndrome live vaccines, the effect of preventing and controlling the highly pathogenic porcine reproductive and respiratory syndrome is still debated by academia, and meanwhile, the pathogenesis of many epidemic diseases is difficult to find, so that the development of the vaccines is difficult, such as African swine fever virus, although the research on the vaccine against the epidemic diseases in various countries around the world is fatally stricken, the research on the vaccine against the epidemic diseases is not overcome all the day. The antiviral medicine for treatment can directly inhibit and kill viruses, has more using methods, can be injected, fed in a mixed way and drunk in a mixed way, but has the defects that the normal metabolism of cells is damaged while the viruses are inhibited and killed, serious medicine residue exists, the health of human beings can be directly harmed, and the viruses can generate medicine-resistant variant strains to the medicines, so that the medicine effect is reduced or lost. In summary, in the face of sudden animal epidemic diseases, the current prevention means and treatment measures cannot really and economically control the development of epidemic diseases of domestic livestock and poultry, so that an effective prevention and treatment measure or treatment method is urgently needed.

Interferon (IFN) is a glycoprotein with high activity and multiple functions produced by macrophages, lymphocytes and somatic cells after human and animal cells are infected by viruses or stimulated by Interferon inducers such as nucleic acid, bacterial endotoxin, mitogen and the like. Can be detected in spleen, liver and peripheral blood lymphocyte of normal organism. The protein has multiple biological activities of broad-spectrum antivirus, antitumor, immunity regulation and the like, and is an ideal antivirus biological agent. Interferons can be divided into three major classes according to their source, structure and function: type I, type II and type III. The type I interferon is mainly produced by leucocytes and fibroblasts, has the main antiviral effect and the auxiliary immune enhancement effect. Mainly including IFN-alpha, IFN-beta, IFN-omega, it is mainly interference virus DNA or RNA synthesis. Type II interferons are produced by activated T cells and NK cells, and have a primary role in immune enhancement and an auxiliary role in antiviral action, and mainly comprise IFN-gamma. The type III interferon is generated by epithelial cells and mucous membrane cells, has similar functional action to the type I interferon, mainly has antiviral effect, is mainly IFN-lambda, is divided into three subtypes of lambda 1, lambda 2 and lambda 3 by human beings, and is only separated into two subtypes of lambda 1 and lambda 3 at present.

At present, the research on the biological functions and action mechanisms of INF-alpha, beta and gamma is reported more, and the research on type III is reported less. The main biological functions of interferons can be summarized as: 1. broad-spectrum antiviral function: the I type and II type interferon genes can be expressed by the activation of an inducer, the transcription of the interferon inducer gene is activated by an expression product through a specific signal transduction path, and a plurality of anti-virus enzymes and proteins with the function of blocking virus replication are synthesized by an organism to resist the infection of the virus to organism cells; 2. the immune regulation function is as follows: type I interferons enhance MHC-class I molecule expression while inhibiting MHC-class II molecule expression; the II type interferon can promote the expression of MHC-II type molecules and the synergistic regulation of the two types of interferons to ensure that the organism is in the optimal immune response state. 3. The immune enhancement function is as follows: both type I and type II interferons can stimulate NK cells and enhance the killing function of the NK cells, and are beneficial to eliminating virus infection of organisms. Type III interferons (also known as IFN- λ s or IL-28/29) are a new class discovered in 2003 and structurally and genetically similar to members of the IL-10 family, but exhibit type I IFN-like activity, exerting antiviral, antitumor and immunomodulatory effects primarily through the JAK-STAT signaling pathway. IFN- λ s receptor expression is primarily in epithelial cells, including epidermal, respiratory and gastrointestinal epithelial cells. Type III interferons cause relatively minor side effects due to the targeting advantage of their receptors (IFN- λ s) relative to type I interferons. In addition, IFN- λ s are important mediators of the antiviral response in mucosal, epithelial tissues, and are critical for protection of GI epithelium, possibly as potential antiviral therapeutics targeting mucosal infections, and also play an important role in mucosal immunity. Overall, type III interferons have a lower ability to induce expression of ISGs than type I interferons. Many in vivo experiments have shown that type I and type III interferons are almost absent during the course of the in vivo antiviral response. Since some cells infected with the virus fail to respond to type III interferon, the IFN-human anti-viral system alone does not provide complete protection against systemic viral infection, and type I interferon is essential in response to systemic viral infection. Type III interferons have a unique role in the gastrointestinal epithelial cell antiviral response, and this role does not overlap with type I interferons.

The CD40L molecule is a type II transmembrane glycoprotein and is a member of Tumor Necrosis Factor (TNF) superfamily, CD40L is mainly expressed in activated CD4+ T lymphocytes, and partially activated CD8+ T cells, basophils, mast cells and NK cells are also expressed. The CD3 monoclonal antibody can stimulate the expression of CD40L by T cells. The interaction between CD40L and CD40 molecules has been shown to promote the activation, proliferation and antibody production of T and B lymphocytes. Meanwhile, CD40L has specific immunoregulation function on various immune cells, so that the vaccine is widely applied to vaccine development. Numerous studies have shown that CD40-CD 40L-mediated signaling can induce activation of Antigen Presenting Cells (APC). APC activation provides an antigen-stimulating signal and a second signal that act synergistically to initiate an immune cascade that promotes humoral and cellular immune functions. In the thymus, CD40 is expressed on cortical, medullary epithelium and thymic stromal cells. These cells continue to express CD40 at low levels in non-disease states, as opposed to CD40, where CD40L is not expressed. By intramuscular injection of the antiviral composition of the present invention, a CD40-CD40L response can be rapidly initiated, triggering a series of specific immune responses, including: (1) promote the differentiation, development, proliferation and maturation of B cells, and participate in the secretion and type conversion of immunoglobulin; (2) promoting the excitation and the directional differentiation of T cells and exerting the specific killing function of the cells; (3) participate in the differentiation and functional regulation of DCs. The CD40L molecule can interact with CD40 on the surface of APC to activate APC, and promote APC costimulatory molecule expression and cytokine secretion.

In a specific immune response, specific CTL firing and expansion must first be initiated by the interaction of Th cells with APC (DC), and the Th-fired DC will then transmit a signal to the CTL, where the DC acts as a bridge between the Th cells and the CTL. However, recent studies have found that priming of CTLs can bypass Th cells by priming the CD40 molecule of DCs. The lack of CD4+ T cell help has been reported in foreign literature to not affect CD40 dependent CD8+ T cell responses, indicating that other cells provide expression of CD40L to activate antigen presenting cells. Because CD8+ T cells are capable of expressing CD40L, they are able to interact directly with APCs, providing assistance for self-activation.

In summary, the antiviral effects of type I and type III interferons are non-specific and can provide a wide range of antiviral effects to the animal body. Meanwhile, the CD40L co-agonist is assisted to induce and generate various cytokines, activate APC, specifically promote the presentation of various virus antigens and improve the level of cellular immunity and humoral immunity.

Disclosure of Invention

The present invention is directed to solving, to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide an antiviral composition, a preparation method and an application thereof, which can be used for actually preventing or treating animal, especially swine viral plague, significantly improving immune response of immune animals, including humoral and cellular immune response, and improving antibody level, thereby improving immune effect of vaccines, and simultaneously shortening immune window period and prolonging protection period.

The invention aims to provide an antiviral composition, which comprises a liquid composition mainly taking porcine alpha interferon, porcine lambda 3 interferon and porcine CD40L as active units and taking arginine, glycerol and the like as protective agents.

According to a specific embodiment of the present invention, in the above antiviral composition, the porcine interferon-alpha encoding gene is preferably cloned and linked to a recombinant expression vector for expression.

According to a specific embodiment of the present invention, in the above antiviral composition, the λ 3 interferon encoding gene is preferably cloned and ligated to a recombinant expression vector for expression.

According to a specific embodiment of the present invention, in the above antiviral composition, the porcine CD40L encoding gene is preferably cloned and ligated to a recombinant expression vector for expression.

Preferably, the recombinant expression vector is a prokaryotic expression vector, more preferably a pET series expression vector is used, such as pET28 and pET 32.

According to the specific embodiment of the invention, the coding gene of the recombinant porcine alpha interferon is Seq ID No: 1, and (b) is shown in the specification.

According to the specific embodiment of the invention, the encoding gene of the recombinant porcine lambda 3 interferon is Seq ID No: 2.

According to a specific embodiment of the invention, the coding gene of the recombinant porcine CD40L is Seq ID No: 3, and (b) is shown in the specification.

Preferably, the final concentration of the alpha interferon is 50-500 ten thousand units/ml, the final concentration of the lambda interferon is 0.5-5 ten thousand units/ml, and the final concentration of the CD40L is 10-40 ug/ml; more preferably, the final concentration of the interferon alpha is 100 ten thousand units/ml, the final concentration of the interferon lambda 3 is 1 ten thousand units/ml, and the final concentration of the CD40L is 20 ug/ml.

According to a specific embodiment of the present invention, the protective agent used in the antiviral composition is composed of arginine, glycerol, sucrose, mannitol.

Preferably, the final concentration of arginine is 0.02-0.1 mol/L, the final concentration of glycerol is 1% -5%, the final concentration of mannitol is 0.05-0.2 mol/L, and the final concentration of sucrose is 1% -3%; more preferably, the final concentration of arginine is 0.05mol/L, the final concentration of glycerol is 5%, the final concentration of mannitol is 0.1mol/L, and the final concentration of sucrose is 1.5%.

According to a specific embodiment of the present invention, the antiviral composition of the present invention is prepared by a method comprising the steps of:

1) weighing the components of the protective agent in proportion, completely dissolving, and adjusting the pH value to 7.2;

2) the biological activity of the prepared alpha interferon and the prepared lambda 3 interferon is measured, and the alpha interferon and the lambda 3 interferon are added into the protective agent according to the activity unit obtained by measurement;

3) measuring the protein concentration of the prepared CP40L recombinant protein, and adding the protein concentration into the protective agent in proportion;

preferably, the method of determining the protein concentration is the BCA method.

4) And (3) carrying out sterile filtration and subpackaging on the protective agent containing the porcine alpha interferon, the porcine lambda 3 interferon and the porcine CD40L recombinant protein, and storing at 2-8 ℃.

According to the specific embodiment of the invention, the application of the antiviral composition in resisting viruses such as epidemic diarrhea and porcine pseudorabies in animals is included.

According to a particular embodiment of the invention, the antiviral application of the viral composition is by intramuscular injection.

Preferably, the injection dosage is 0.5-2 ml, more preferably, 0.5ml is injected into piglets and 2ml is injected into bred pigs and sows.

Preferably, the animal is a mouse, rat, rabbit, dog, pig, cow, more preferably the animal is a pig.

According to a particular embodiment of the invention, there is provided a method of raising an immune response, in particular a cellular immune response, in an animal, characterised in that an antiviral composition as described above is administered to the animal.

Preferably, the animal is a mouse, rat, rabbit, dog, pig, cow.

According to an embodiment of the present invention, there is provided a vaccine comprising the antiviral composition, wherein the vaccine is a whole virus inactivated vaccine or a subunit vaccine, and the antiviral composition is used as an immunological adjuvant or an immunopotentiator.

Preferably, the whole virus inactivated vaccine includes, but is not limited to, porcine pseudorabies inactivated vaccine, porcine epidemic diarrhea inactivated vaccine, porcine circovirus inactivated vaccine, porcine parvovirus inactivated vaccine, foot-and-mouth disease inactivated vaccine, and the like; more preferably a porcine pseudorabies virus inactivated vaccine.

Preferably, the subunit vaccine includes, but is not limited to, subunit vaccines prepared by recombining virus such as African swine fever, porcine epidemic diarrhea, porcine circovirus by genetic engineering technology.

Compared with the prior art, the invention has the advantages and positive effects that: the porcine alpha interferon and the porcine lambda 3 interferon are both gene engineering recombinant protein products, the biological activity is 1-3 orders of magnitude higher than that of natural interferon, the porcine alpha interferon and the natural interferon both have obvious antiviral activity, and the addition of the porcine lambda 3 interferon can make up the characteristic that the porcine alpha interferon has insufficient antiviral effect in epithelial cells and mucous membrane tissues. CD40L is a receptor of an auxiliary stimulation factor CD40, and the combination of the two can not only promote the differentiation of T cells, but also play a role in killing cells specifically and indirectly further improving the antiviral effect; it also promotes B cell activation and humoral immune response.

Drawings

FIG. 1 shows the effect of recombinant protein purification

M: is a protein Marker; 1 is recombinant porcine IFN-alpha; 2 is recombinant porcine IFN-lambda 3; 3 is recombinant porcine CD 40L.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

EXAMPLE 1 construction of prokaryotic expression vectors

The total RNA of the pig liver is extracted by a Trizol method, and cDNA is synthesized by a reverse transcription kit by taking the total RNA as a template. According to the sequences of IFN-alpha, IFN-lambda 3 and CD40L of pigs published by Genebank, an amplification primer with BamH I and Xho I enzyme cutting sites is designed, PCR amplification is carried out, the obtained product is the corresponding target gene sequence, see Seq ID No.1, Seq ID No.2 and Seq ID No.3, and after agarose gel electrophoresis identification, a T vector (pUC-19) is connected, identified and sent to a sequencing company for sequencing.

And carrying out double enzyme digestion on the T vector containing the target gene and pET32a with correct sequencing by using BamH I and Xho I enzymes respectively, carrying out agarose gel electrophoresis identification on the digestion products, connecting the target gene identified to be correct with the pET32a vector, and transforming DH5a bacteria. Selecting a monoclonal, inoculating the monoclonal into 5ml LB liquid culture medium containing ampicillin sodium with the final concentration of 100ug/ml, culturing at 37 ℃ and 180rpm with shaking for 12h, extracting pET 32-IFN-alpha, pET 32-IFN-lambda 3 and pET32-CD40L plasmids respectively by using a complete gold Plasmid mini kit I kit, sending the plasmids to a sequencing company for sequencing, and converting the plasmids with correct sequencing into plasmidsTransetta (DE3) was plated on LB solid agar plates and incubated overnight at 37 ℃.

Picking a single colony on an LB solid plate, inoculating the single colony into 5ml of LB liquid culture medium containing ampicillin sodium with the final concentration of 100 mu g/ml, carrying out shake culture at 37 ℃ and 180rpm for 10h, adding glycerol with the final concentration of 30 percent, and preserving at-80 ℃ to obtain recombinant prokaryotic expression vector strains of the porcine IFN-alpha, the IFN-lambda 3 and the CD40L respectively.

EXAMPLE 2 fermentation culture and purification of recombinant proteins

1. First-stage seed propagation and identification (rejuvenation) a proper amount of glycerol strain is taken by an inoculating ring hook and inoculated on an LB agar plate containing 100 mu g/ml ampicillin sodium, the culture is carried out for 12 hours at 37 ℃, at least 5 monoclonal colonies are picked, and PCR detection verification is carried out. And (4) storing the qualified colony plate at 2-8 ℃, wherein the subculture does not exceed 5 generations after 14 days.

2. Secondary seed propagation the primary seeds were inoculated into LB liquid medium (50 mL/250 mL) containing 100. mu.g/mL ampicillin sodium, and cultured at 37 ℃ for 10 hours to give secondary seeds (seeds for production).

3. Three-stage seed culture the secondary seeds were inoculated into LB liquid medium (250 mL/1000mL, two flasks) containing 100. mu.g/mL ampicillin sodium, and cultured at 37 ℃ for 10 hours to give three-stage seeds (seeds for production).

4. Adding the prepared fermentation culture solution into a fermentation tank before fermentation, simultaneously correcting a pH electrode and an oxygen dissolving electrode, and then putting the whole tank into a sterilization pot for sterilization, wherein the sterilization conditions are as follows: 121 ℃ and 20 min. Taking out the mixture when the mixture is hot after the sterilization is finished.

5. Inoculating three-stage seeds in 10% of the total volume of the fermentation liquid, adding 1% sterile glucose, adjusting pH to 6.8 after inoculation, and maintaining the dissolved oxygen at above 30% at 37 deg.C under the linkage of rotation speed and dissolved oxygen.

6. Culturing the induced expression bacteria liquid for 6-10 hours, adding IPTG 0.5mmol/L when the OD value of the bacteria body can reach more than 2.5, adjusting the temperature to 28 ℃, and stopping culturing after inducing for 12 hours.

7. And (2) combining collected thalli, putting the combined thalli into a cleaned centrifugal tube, weighing and balancing, centrifuging for 20 minutes at 4 ℃ by using a high-speed freezing centrifuge at 10000r/min, pouring off a supernatant, resuspending and dissolving a precipitate by using 1/5 original volume of Tris-HCl (0.02 mol/L Tris, 0.5mol/L NaCl, 10% glycerol, adjusting the pH value to 7.9 by using concentrated hydrochloric acid, filtering by using 0.45 mu m) buffer solution, weighing and balancing, centrifuging again, and continuously washing twice by using the method. And finally, pouring out the supernatant, weighing the precipitate, adding 1.0-5.0 ml of Tris-HCl buffer solution into 100mg of the thallus precipitate, and suspending and dissolving. Adding a protease inhibitor into the resuspended and dissolved thallus according to the proportion of 1:100, carrying out ultrasonic lysis under ice bath conditions, working for 5 seconds, resting for 6 seconds, continuously carrying out ultrasonic treatment for 40 minutes, centrifuging at 10000r/min and 4 ℃ for 20 minutes, collecting supernate, and filtering with a 0.45-micrometer filter membrane.

8. Purification recombinant proteins were purified using affinity chromatography and an APPS 50D protein purification system: connecting an agarose gel prepacked column (kept at 4 ℃) to a detector connecting tube, opening a purification software, and performing connection detection; putting the liquid inlet A pipe into deionized water, setting the flow rate at 2.0ml/min, running for 25 minutes, and washing the ethanol in the pre-packed column; and taking the feed liquid A out of the deionized water, putting the feed liquid A into the balance combined liquid, setting the flow rate to be 2.0ml/min, operating for 25 minutes, and carrying out column balance. And taking the liquid inlet pipe A out of the equilibrium binding solution, putting the liquid inlet pipe A into the sample liquid to be purified, setting the flow rate to be 0.5ml/min, and operating. After the sample is fed, the feed liquor pipe A is put into the mixed protein washing liquid at the flow rate of 2.0ml/min, and the operation is carried out for 100 minutes (if the base line does not tend to be stable, the time can be prolonged). And after washing of the hybrid protein is finished, putting the liquid inlet A tube into the target protein eluent at the flow rate of 1.0ml/min, eluting the target protein, starting to collect the target protein after a protein peak appears, stopping collection after the protein peak falls, and marking.

9. Endotoxin removal

Toxin Eraser from Nanjing Kingsri^TMEndotoxin Removal by Endotoxin Removal Resin: firstly, the pH value of a sample is adjusted to 7.5 by using 0.1M hydrochloric acid, then the sample is added into a gel column which is balanced by using a balance liquid, the flow rate is controlled to be 0.2ml/min, and a flow-through liquid is collected.

Example 3 quality testing of recombinant purified proteins

1. Determination of purity

The purity of the recombinant protein was determined by SDS-PAGE electrophoresis in combination with Bio-Rad Image Lab software. The sample loading amount is not less than 1ug, after SDS-PAGE electrophoresis, dyeing and decoloring, only a band which is consistent with the molecular weight of the target protein is on the electrophoresis gel, no obvious hybrid protein band is seen, the purity of the analysis of Image Lab software is more than 90.0 percent, and the determination result is shown in figure 1.

2. Determination of biological Activity

Referring to appendix of Chinese pharmacopoeia (2010 version) 'interferon biological activity assay (cytopathic effect inhibition method)', MDBK cells and VSV viruses are used as evaluation systems to determine the biological activities of purified recombinant proteins IFN-alpha and IFN-lambda 3. The biological activities of the porcine alpha and lambda 3 interferons are 2.3X 10 respectively^9.0U/ml and 1.8X 10^5.0U/ml。

3. Protein content determination

The protein content of three recombinant purified proteins is detected by using a BCA method protein quantitative detection kit of Kangji century company, wherein IFN-alpha, IFN-lambda 3 and CD40L are respectively 0.6mg/ml, 0.4mg/ml and 0.4 mg/ml.

EXAMPLE 4 preparation of protective and antiviral compositions

(1) Tris buffer solution is used for preparing solution containing arginine with final concentration of 0.2mol/L, glycerol 20%, mannitol 0.4mol/L and trehalose 6%, and after complete dissolution, concentrated hydrochloric acid is used for adjusting the pH value to 7.2.

(2) Diluting the porcine alpha interferon and the porcine lambda interferon to 400 ten thousand units/ml and 4 ten thousand units/ml respectively by using a Tris buffer solution, and then mixing the diluted porcine alpha interferon and the porcine lambda interferon with the same volume of a protective agent;

(3) diluting the CD40L recombinant protein to 40ug/ml with Tris buffer solution, and mixing with the mixture of interferon and protective agent in equal volume;

(4) adjusting pH to 7.2 with concentrated hydrochloric acid, and sterile filtering to obtain antiviral composition mixed solution.

Example 5 antiviral composition safety assay

1. Mouse method

By adopting a mouse test method, 5 mice are used for each batch of samples, and each mouse is weighed before injection and should be 18-22 g. Each mouse was injected with 0.5ml of the test article intraperitoneally and observed for 7 days. In the observation period, all mice are kept healthy, no abnormal reaction occurs, and the weight of each mouse is increased when the mice are expired.

2. Endotoxin assay

The endotoxin content in the antiviral composition sample is detected by adopting a ToxinSensor ™ gel-method endotoxin detection kit of Nanjing Kingsrey Bio-corporation, and the detection result shows that the endotoxin content in the composition does not exceed 20 EU/ml.

Example 6 verification of the Effect of the target animal against porcine epidemic diarrhea Virus

15 piglets (long white pigs) with negative epidemic diarrhea virus antigen (fluorescent quantitative PCR method) and antibody (cell neutralization antibody titer is not higher than 1: 4) of 4-day-old pigs are selected and divided into 3 groups, wherein the 1 st and 2 th groups are test groups, and the 4 th group is a control group, and each group comprises 5 piglets. On the 1 st to 6 th days of the experiment, the 5 th piglets in the 1 st group are respectively injected with 0.5 ml/head of porcine recombinant IFN-alpha (100 ten thousand units) in the morning and at night, and the 5 th piglets in the 2 nd group are respectively injected with 0.5 ml/head of antiviral composition in the morning and at night; on day 3 of the experiment, 1000TCID was orally administered to each of the test group and the control group₅₀The porcine epidemic diarrhea virus attack virus; clinical observation is carried out for 7 days after the toxin is attacked, clinical symptoms of diarrhea, vomiting and the like of each group of pigs are observed and recorded every day, and the porcine epidemic diarrhea virus in the excrement is measured by a fluorescent quantitative PCR method. The test results are shown in Table 1.

TABLE 1 statistical table of clinical manifestations and test results of test and control groups after challenge

Note: the fluorescence quantitative method is a test group self-construction method, and the positive result is obtained when the CT value is lower than 37.

The experimental result shows that compared with a control group for attacking, the morbidity and the mortality of the attacking pig can be reduced by singly using the IFN-alpha and the antiviral composition before attacking, wherein the morbidity and the mortality are respectively reduced by 20% and 25% by singly using the IFN-alpha, and the morbidity and the mortality are respectively reduced by 40% (more transient diarrhea) and 50% by using the antiviral composition; meanwhile, the experimental result also shows that the antiviral composition used before infection can delay the disease onset time and obviously reduce the toxin expelling amount. In the latent period of the onset of disease, the generation of antiviral factors of organisms can be started after the interferon is used, a network protection mechanism is formed, antiviral protein factors are released, and the immune level of the pig body can be effectively improved. Therefore, the antiviral composition has a good prevention and treatment effect on porcine epidemic diarrhea.

Example 7 use as an immunopotentiator in inactivated porcine pseudorabies vaccine (mouse)

The virus titer in vero cells was 10^7.8TCID₅₀A/ml porcine pseudorabies virus gI/gE deletion strain virus liquid is inactivated and then mixed with an antiviral culture in a ratio of 2:1, and then mixed with a conventional oil adjuvant in a ratio, and then inoculated with 8 Kunming mice of 18-22 g to form a test group A, and meanwhile, a vaccine test group B and a blank control group C which are only inoculated with the porcine pseudorabies virus and the conventional oil adjuvant are arranged. On days 14 and 28 after inoculation, serum was collected and separated, and the neutralizing antibody titer and IFN-. gamma.content in the serum were measured by cell neutralization assay and ELISA, respectively. The specific results are shown in Table 2. As can be seen from the results, the antiviral composition can obviously enhance the neutralizing antibody titer and the cellular immunity level of the inactivated porcine pseudorabies vaccine after being used as an immunopotentiator.

TABLE 2 statistical table of the results of the measurement of serum neutralizing antibody titer and IFN-. gamma.content

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<110> Beijing great Weijia Biotech Ltd

<120> an antiviral composition, a preparing method thereof and applications of the same

<130> 1

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 423

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgcgacctgc ctcagaccca cagcctggct cacaccaggg ccctgaggct cctggcacaa 60

atgaggagaa tctctccctt ctcctgcctg gaccacagaa gggactttgg atttccccaa 120

gaggccttgg ggggcaacca ggtccagaag gctcaagcca tggctctggt gcatgagatg 180

ctccagcaga ccttccagct cttcagcaca gagggctcgg ctgctgcctg ggatgagagc 240

ctcctgcacc agttctacac tggactggat cagcagctca gggacctgga agcctgtgtc 300

atgcaggagg cggggctgga agggaccccc ctgctggagg aggactccat cctggctgtg 360

aggaaatact tccacagact caccctctat ctgcaagaga agagctacag cccctgtgcc 420

tgg 423

<210> 2

<211> 519

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtgcctgtcc ctgaagccct cagggccctc ccaggagcaa ggggctgcca cttggcccag 60

ttcaagtctc tgtccccaca agcgctgcag gccttcaaga gggccaagga tgcctttgaa 120

gagtccctct tggaggactg gaactgcagc tcccgcatct tccccaggag cagggacctg 180

aagcagctgc aggtgtggga gcgccccgtg gccttggagg ccgaggtggc cctgaccctc 240

agcgtcctgg gctccttggc gaactcatcc ctgcacagca gcctggacca gccccttcac 300

acgctgcgcc acatccacgc ccagctccag gcctgtgtcc cagctcagcc catggcaggc 360

ccccggcccc ggggccgcct ccaccactgg ctgcaccggc tccaggaggc ccagaagaag 420

gagccccaga gctgcctgga agcctctgtc atgttcaacc tcttccgcct cctcacccgg 480

gacctgaaat gtgtcgccag tggagacctg tgtgtctga 519

<210> 3

<211> 756

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgatcgaaa cgtacagcca accttcgccc cgctctgtgg ccgctggacc acccgtcagt 60

atgaaaatct ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120

ctttttgcag cgtaccttca cagaagattg gacaagatag aagatgaaag gaatcttcat 180

gaagattttg tgttcataaa aacgatacag agatgcaagc aaggagaggg gtccttatcc 240

ttattgaact gtgaggaaat cagaagccag tttgaagacc tggtcaaggg tataatgcaa 300

agcaaagaag tgaagaagaa agaaaaaagc tttgaaatgc acaaaggcga tcaggatcct 360

caaattgcgg cacatgtcat aagcgaggcc agtagtaaaa cagcatctgt cctgcagtgg 420

gcccccaaag ggtactacac cctcagcacc aacttggtga ccctggaaaa cgggagacag 480

ctggccgtca aaagacaagg aatctattac atctacgccc aagtcacctt ctgctccaac 540

cgggacgccg cgggtcaagc tcccttcata gccagcctct gcctgaggtc cccaagcggg 600

tcggagagaa tcttactccg cgcggccaac acccacagtt cctccaagcc ctgcgggcag 660

caatccattc acttgggcgg agtcttcgag ttgcaacccg gcgcttcggt gttcgtcaac 720

gtgactgatc caagccaagt gagccacggg acctga 756

Claims

1. An antiviral composition, which is characterized by being prepared by combining porcine alpha interferon, porcine lambda 3 interferon, porcine CD40L protein and a protective agent.

2. The antiviral composition according to claim 1,

the porcine alpha interferon is obtained by cloning and connecting a porcine alpha interferon coding gene to a recombinant expression vector for expression;

the porcine lambda 3 interferon is obtained by cloning and connecting a porcine lambda 3 interferon encoding gene to a recombinant expression vector for expression;

the porcine CD40L is obtained by cloning and connecting a porcine CD40L encoding gene to a recombinant expression vector for expression;

optionally, the recombinant expression vector comprises a prokaryotic expression vector, a yeast expression vector and a eukaryotic expression vector;

optionally, prokaryotic expression vectors include, but are not limited to, pET series vectors, pQE series vectors, pGEX series vectors, pMAL series vectors, pBV series vectors, and the like, preferably pET series vectors and pBV series vectors, more preferably pET28, pET32, and pBV 220;

optionally, eukaryotic expression vectors include, but are not limited to, pcDNA series vectors, pCMV series vectors, and pEF series vectors, preferably pcDNA series vectors are used, more preferably pcdna3.1 vectors are used;

the porcine alpha interferon coding gene is Seq ID No: 1;

the porcine lambda 3 interferon coding gene is Seq ID No: 2;

the porcine CD40L coding gene is Seq ID No: 3, and (b) is a sequence shown in the specification;

optionally, the final concentration of the porcine alpha interferon is 50-500 ten thousand units/ml, the final concentration of the porcine lambda 3 interferon is 0.5-5 ten thousand units/ml, and the final concentration of the porcine CD40L is 10-40 ug/ml; more preferably, the final concentration of the porcine interferon-alpha is 100 ten thousand units/ml, the final concentration of the porcine interferon-lambda 3 is 1 ten thousand units/ml, and the final concentration of the porcine CD40L is 20 ug/ml;

optionally, the porcine alpha interferon, the porcine lambda 3 interferon and the porcine CD40L prokaryotic recombinant protein are subjected to endotoxin removal by a two-phase extraction method or affinity chromatography method or ion exchange method or ultrafiltration method; more preferably, the method for removing endotoxin is ultrafiltration.

3. The antiviral composition according to claim 1, wherein said protective agent is composed of arginine, glycerol, trehalose, mannitol;

optionally, the final concentration of arginine is 0.02-0.1 mol/L, the final concentration of glycerol is 1% -5%, the final concentration of mannitol is 0.05-0.2 mol/L, and the final concentration of sucrose is 1% -3%; more preferably, the final concentration of arginine is 0.05mol/L, the final concentration of glycerol is 5%, the final concentration of mannitol is 0.1mol/L, and the final concentration of trehalose is 1.5%.

4. A method of preparing the antiviral composition of claims 1-3, comprising:

1) weighing the components of the protective agent according to a proportion of 4 times of the final concentration, completely dissolving, and adjusting the pH value to 7.2;

2) the prepared porcine alpha interferon and porcine lambda 3 interferon are measured for biological activity, and are properly diluted by Tris buffer solution according to the activity unit obtained by measurement and then are mixed with a protective agent in equal volume;

3) measuring the protein concentration of the prepared porcine CD40L recombinant protein, and adding the protein concentration into the protective agent in proportion;

optionally, methods for determining protein concentration include Coomassie Brilliant blue method (Bradford), Folin-phenol reagent method (Lowry), BCA method, etc.;

5. Use of an antiviral composition as claimed in any of the claims 1 to 3 for combating viruses in animals, wherein the antiviral composition is used for increasing the immune response, in particular the cellular immune response, of an animal;

the animal is mouse, rat, rabbit, dog, pig, or cattle.