TW201945021A - Composition for the prevention of mycoplasma synoviae infection - Google Patents

Abstract

The present invention is related to a composition for the prevention of Mycoplasma synoviae infection, comprising NOX, MS53-0285 or the combination thereof as active ingredients. The present composition exhibits effects in relieving the symptoms caused by Mycoplasma synoviae infection and can be served as a useful tool for the prevention and control of poultry disease.

Description

Composition for preventing and controlling poultry synovial mold mold infection

The present invention relates to a composition for preventing and controlling mycoplasma infection; in particular, it relates to a composition for preventing and controlling poultry synovial mycoplasma infection.

Mycoplasma spp. Is the smallest prokaryotic microorganism currently known to be able to replicate outside the cell, ranging in size from a spherical shape with a diameter ranging from 0.3 μm to 0.9 μm to a filamentous shape that can be as long as 1 μm. In terms of nutritional requirements, mycelium is a fastidious bacteria, which is difficult to cultivate and grows very slowly. Several species of mycoplasma are known to be associated with a variety of human or animal diseases.

According to literature reports, there are dozens of mycoplasmas that can infect poultry. Among them, Mycoplasma synoviae is listed in the World Organisation for Animal Health (OIE) poultry mycoplasmosis file. Pathogenic microorganisms, showing its importance for the prevention and control of poultry diseases.

Synovial mycoplasma in poultry is also associated with the occurrence of synovitis or arthritis. Infecting poultry synovial mold on broiler chickens will result in poor feed efficiency, higher mortality rates, and higher slaughter quality. After breeder or laying hens become infected, egg production rates and embryo mortality will increase. . After chickens are infected with mycoplasma, they are more likely to cause secondary infections of other viral or bacterial pathogens, causing huge economic losses to chicken farmers. Although global animal vaccine manufacturers have developed live and dead bacteria vaccines for use in the chicken industry, mold mold is not easy to cultivate and the cost of culture is high. Therefore, there is still a need in the field for vaccines against poultry synovial mold infection. .

It is an object of the present invention to provide an antigen suitable for a poultry synovial mold subunit vaccine, and to prepare a subunit vaccine based on it, so as to reduce the cost of epidemic prevention.

Another object of the present invention is to provide a subunit vaccine containing multiple antigens for preventing and controlling poultry synovial mycoplasma infection, which has an immunoprotective effect superior to a single antigen vaccine.

In order to achieve the above object, the present invention provides a composition for preventing and controlling synovial mycoplasma infection in poultry, which comprises: an active ingredient comprising Tuf, NOX, MS53-0285, or a combination thereof; wherein the aforementioned Tuf has SEQ ID NO: 01, the aforementioned NOX has the sequence shown in SEQ ID NO: 02, the aforementioned MS53-0285 has the sequence shown in SEQ ID NO: 03; and a pharmaceutically acceptable adjuvant.

Preferably, the aforementioned active ingredient is selected from at least two of the group consisting of the following proteins: Tuf, NOX and MS53-0285. More preferably, the aforementioned active ingredients include Tuf, NOX, and MS53-0285.

Preferably, the concentration of the aforementioned active ingredient is 40 to 900 μg / mL, which is based on the total volume of the aforementioned composition.

Feasibly, the aforementioned pharmaceutically acceptable adjuvant is: Freund's complete or incomplete adjuvant, aluminum gel, surfactant, polyanion, peptide, oil emulsion, or a combination thereof.

Preferably, the aforementioned composition further comprises a pharmaceutically acceptable additive. Feasibly, the aforementioned pharmaceutically acceptable additives are solvents, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, or combinations thereof.

Preferably, the aforementioned symptoms caused by the infection of the synovial mycoplasma of poultry are tracheal lesions, balloon lesions, arthritis, or a combination thereof.

Preferably, when the aforementioned active ingredient comprises Tuf, the aforementioned symptoms caused by the infection of the poultry synovial mold are at least arthritis. Preferably, when the aforementioned active ingredient comprises NOX, MS53-0285, or a combination thereof, the aforementioned symptoms caused by the infection of the synovial mold of the poultry are at least airbag lesions. Preferably, when the aforementioned active ingredient comprises MS53-0285, the aforementioned symptoms caused by the infection of synovial mold of poultry are at least tracheal lesions.

Preferably, when the aforementioned active ingredients include Tuf, NOX, and MS53-0285, the aforementioned symptoms caused by the infection of synovial mold of poultry are tracheal lesions, balloon lesions, and arthritis.

The present invention further provides a performance vector, comprising: a nucleotide sequence having a sequence shown in SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, or a combination thereof; a performance element, comprising A promoter and a ribosome binding site; and a fusion partner sequence.

The present invention further provides a performance vector, which comprises: a nucleotide sequence that is translated into a protein of Tuf, NOX, MS53-0285, or a combination thereof; wherein the aforementioned Tuf comprises the sequence shown in SEQ ID NO: 01 The aforementioned NOX includes the sequence shown in SEQ ID NO: 02, and the aforementioned MS53-0285 includes the sequence shown in SEQ ID NO: 03; a performance element including a promoter and a ribosome binding site; and a fusion partner sequence.

Preferably, the aforementioned fusion partner is DsbC of E. coli, MsyB of E. coli, FklB of E. coli, or a combination thereof.

Preferably, the aforementioned expression vector has a sequence shown in SEQ ID NO: 07, SEQ ID NO: 08, or EQ ID NO: 09.

The present invention further provides the use of a protein for preparing a composition for preventing and controlling poultry synovial fungal infection, wherein the aforementioned protein comprises Tuf, NOX, MS53-0285, or a combination thereof; wherein the aforementioned Tuf has the sequence shown in SEQ ID NO: 01 For the sequence, the aforementioned NOX has the sequence shown in SEQ ID NO: 02, and the aforementioned MS53-0285 has the sequence shown in SEQ ID NO: 03.

In summary, the present invention discloses an antigen for controlling poultry synovial mold infection, which can be used as an active ingredient of a subunit vaccine. The research and development achievements of the present invention not only provide new options for poultry synovial mold prevention, but also reveal that a "cocktail" subunit vaccine combining two or more antigens (that is, containing two or more antigens as active ingredients) has stronger immunity. Induced effect.

The research and development results of the present invention confirm that Tuf, NOX, and MS53-0285 can be used as active ingredients of a secondary unit vaccine for preventing and controlling poultry synovial mold infection. The “prevention of synovial mycoplasma infection of poultry” in the present invention refers to reducing the degree of infection of poultry synovial mycoplasma by poultry; for example, reducing the symptoms caused by the infection of poultry synovial mycoplasma. The aforementioned symptoms include, but are not limited to, tracheal lesions, balloon lesions, or arthritis (eg, swelling of the foot pad, inflammation of the foot pad, etc.). From a scientific point of view, it is impossible to confirm whether the target individual is completely free of any indicated pathogen infection. Therefore, those with ordinary knowledge in the field should understand that in the field of disease control, the so-called "prevention of infection" does not mean that the target individual is not completely infected by any of the indicated pathogens.

One aspect of the present invention is directed to a composition for controlling infection of synovial mycoplasma in poultry. In a preferred embodiment of the present invention, the aforementioned composition is a single-unit vaccine. In a preferred embodiment of the present invention, the composition includes Tuf as an active ingredient, and the aforementioned Tuf has a sequence shown in SEQ ID NO: 01. In another preferred embodiment of the present invention, the composition includes NOX as an active ingredient, and the aforementioned NOX has a sequence shown in SEQ ID NO: 02. In another preferred embodiment of the present invention, the composition includes MS53-0285 as an active ingredient, and the aforementioned MS53-0285 has a sequence shown in SEQ ID NO: 03.

Those with ordinary knowledge in the art should understand that as long as the epitope region of the aforementioned protein is not affected, the aforementioned amino acid sequence can tolerate considerable changes, and still belong to the scope of the present invention. For example, those with ordinary knowledge in the field may change several amino acids in the sequence disclosed before, but still can produce the immune-inducing effect of the present invention; or those with ordinary knowledge in the field may, depending on their needs, Other sequences are added to the previously disclosed sequences to facilitate production, but do not affect the immune-inducing effect referred to in the present invention. The modified sequence in the case of the previous disclosure should still belong to the scope of the present invention.

In one aspect of the present invention, when the aforementioned active ingredient comprises Tuf, the aforementioned symptom caused by the infection of the poultry synovial mold is at least arthritis. However, this does not mean that when the aforementioned active ingredient contains Tuf, it has no effect on other symptoms, but indicates that when the aforementioned active ingredient contains Tuf, it is particularly effective in preventing the symptoms of arthritis.

In one embodiment of the present invention, when the aforementioned active ingredient contains NOX, MS53-0285, or a combination thereof, the aforementioned symptoms caused by the infection of the synovial mycoplasma of poultry are at least balloon lesions. However, this does not mean that when the foregoing active ingredient contains NOX, MS53-0285, or a combination thereof, it has no effect on other symptoms, but indicates that when the foregoing active ingredient contains NOX, MS53-0285, or a combination thereof, the Prevention of symptoms is particularly effective.

In one aspect of the present invention, when the active ingredient comprises MS53-0285, the aforementioned symptoms caused by the infection of the synovial mold of the poultry are at least tracheal lesions. However, this does not mean that when the aforementioned active ingredient contains MS53-0285, it has no effect on other symptoms, but indicates that when the aforementioned active ingredient contains MS53-0285, it is particularly effective in preventing the symptoms of tracheal disease.

In one embodiment of the present invention, when the active ingredients include Tuf, NOX, and MS53-0285, the aforementioned symptoms caused by the infection of synovial mold of poultry are tracheal lesions, balloon lesions, and arthritis.

Another aspect of the present invention is directed to a cocktail vaccine for preventing and controlling poultry synovial mycoplasma infection. The cocktail vaccine mentioned in the present invention means that the vaccine contains two or more kinds of antigens. In a preferred embodiment, the cocktail vaccine referred to in the present invention means that the vaccine contains two or more antigens against the same pathogenic bacteria. In general, combining two or more antigens in a single vaccine does not necessarily have a multiplier effect on the immune-inducing effect of the vaccine. The combination of two or more antigens in a single vaccine may instead cause the two or more antigens to have an adverse effect on their immunity-inducing ability. From the perspective of cost, even if the immune-inducing ability of the two or more antigens does not offset, if it does not have a multiplier effect, it is not worth mixing the two or more antigens in a single vaccine. .

In a preferred embodiment, the active ingredient of the aforementioned cocktail vaccine is selected from at least two of the group consisting of the following proteins: Tuf, NOX, and MS53-0285. More preferably, the aforementioned active ingredients include Tuf, NOX, and MS53-0285. In a feasible implementation form, the active ingredient may be a fusion protein having any two or more of the aforementioned amino acid sequences, as long as the epitope of the amino acid sequence after folding is not destroyed. In another possible implementation aspect, the aforementioned cocktail vaccine uses the aforementioned proteins separately.

In a preferred embodiment, the composition of the present invention further comprises a pharmaceutically acceptable adjuvant and / or a pharmaceutically acceptable additive. The aforementioned pharmaceutically acceptable adjuvant is used to increase the immune effect of the active ingredient, maintain the stability of the active ingredient, and improve the safety of the composition for vaccine use. The aforementioned pharmaceutically acceptable adjuvant may be, but is not limited to, Freund's complete or incomplete adjuvant, aluminum gel, surfactant, polyanion, peptide, oil emulsion, or a combination thereof. The aforementioned pharmaceutically acceptable additives may be, but are not limited to, solvents, stabilizers, diluents, preservatives, antibacterial agents, antifungals, isotonic agents, absorption delaying agents, or combinations thereof.

In a feasible embodiment, the composition of the present invention can be adjusted into an oral administration form, a nasal absorption form, an intravenous injection form, an intramuscular injection form, or an intraperitoneal injection form. In a feasible embodiment, the concentration of the active ingredient in the composition of the present invention is 40 to 900 μg / mL, which is based on the total volume of the foregoing composition. In another feasible embodiment, the concentration of the foregoing active ingredient is 40 to 600 μg / mL, which is based on the total volume of the foregoing composition. In the cocktail vaccine of the present invention, the concentration of the aforementioned active ingredient means the total concentration of all the antigens contained in the cocktail vaccine. For example, in a specific example of the cocktail vaccine of the present invention, the aforementioned Tuf antigen and the aforementioned NOX antigen are used as active ingredients, and the “concentration of the active ingredient” means the sum of the aforementioned Tuf antigen concentration and the aforementioned NOX antigen concentration. For another example, in another specific example of the cocktail vaccine of the present invention, the aforementioned Tuf antigen, the aforementioned NOX antigen, and the aforementioned MS53-0285 antigen are used as active ingredients, and the “concentration of the active ingredient” means the concentration of the aforementioned Tuf antigen, The sum of the concentration of the NOX antigen and the concentration of the MS53-0285 antigen.

On the other hand, one obstacle to the expression of the active ingredient in the prokaryotic expression system is the purification of the active ingredient obtained from the expression of the prokaryotic expression system. Recombinant proteins expressed in a prokaryotic cell expression system are usually not soluble, thus increasing the difficulty and cost of the purification step. In view of this, a feature of the antigen expression vector of the present invention is that it can show a soluble recombinant antigen, thereby simplifying the purification step and its cost.

Therefore, another aspect of the present invention is directed to a performance vector for preparing the aforementioned Tuf, the aforementioned NOX, or the aforementioned MS53-0285. The expression vector of the present invention comprises a nucleotide sequence, a expression element and a fusion partner sequence. The aforementioned nucleotide sequence can be translated into the aforementioned Tuf, NOX, MS53-0285, or a combination thereof.

The aforementioned expression elements refer to the elements required for initiating the transcription and translation process in the expression system. The expression element should include at least a promoter and a ribosome binding site. Preferably, the aforementioned expression element may additionally include: an operator, a transcription / translation enhancer sequence, or a combination thereof. In a preferred embodiment, the expression vector is used in an E. coli expression system, and the expression element can be identified by E. coli.

In a preferred embodiment, in order to make the recombinant protein obtained in a prokaryotic cell expression system soluble, the research of the present invention confirms that DsbC of E. coli, MsyB of E. coli, FklB of E. coli, or a combination thereof is Preferred fusion partners for expressing the aforementioned antigens of the invention. In a preferred embodiment, in order to facilitate the purification step, the expression vector may further include a set of amino-tag sequences (His-tags), a mono-glutamine-S-transferase tag (GST- tag) sequence, or a combination thereof, so that the resulting recombinant protein can be purified by a nickel ion affinity column or a glutamine sulfur affinity column.

Still another aspect of the present invention is directed to the use of the aforementioned Tuf, the aforementioned NOX, or the aforementioned MS53-0285 for the preparation of a composition for controlling a synovial mold infection of poultry. The aforementioned composition for controlling the infection of synovial mycoplasma in poultry is as discussed in the previous paragraph, and will not be repeated here.

The following examples are used to describe the experiments conducted by the present invention to further explain the features and advantages of the present invention. It should be understood that the listed embodiments are merely exemplary illustrations of the claimed invention, and should not be used to limit the scope of patent application of the present invention.

experiment one: Breeding of poultry synovial mold antigen genes.

Design specific primers for different antigen genes (Table 1). Using poultry synovial mold gene body as template, specific primer combination was used to amplify the target gene.

Table 1: Primer pairs used for amplification of target genes.

1. Extraction of poultry synovial mold gene bodies.

Tissue & Cell Genomic DNA Purification kit (GMbiolab, Taiwan) was used to extract the genomic bodies of poultry synovial mold WVU 1853 (American Type Culture Collection ^® 25204 ™). First take 4.5 mL of the culture bacteria solution into a centrifuge tube, and centrifuge (5,870 × g, 5 minutes), then remove the supernatant and collect the bacterial cells. Then, 20 μL of proteinase K (10 mg / mL) and 200 μL of extraction reagent were added and reacted at 56 ° C for 3 hours. After that, 200 μL of a binding solution was added and reacted at 70 ° C for 10 minutes. After the reaction, add 200 μL of absolute alcohol to a microcentrifuge tube and mix uniformly. Pipette all solutions (including sediment) into a spin column, and place the centrifugation tube in a collection tube. . After centrifugation for 2 minutes (17,970 × g), the effluent was decanted and 300 μL of binding reagent was added to the centrifuge tube. After centrifugation for 2 minutes (17,970 × g), the effluent was removed. After that, add 700 μL of wash solution to the centrifuge tube. After centrifugation for 2 minutes (17,970 × g), remove the effluent and repeat the above steps once. Finally, centrifuge at 17,970 × g for 5 minutes to remove residual alcohol. Place the centrifuge tube into a sterilized microcentrifuge tube, and add the appropriate amount of sterile deionized water to drain the genomic DNA.

2. Amplify the target gene by polymerase chain reaction (PCR).

Using poultry synovial mold gene body as template, primers designed for tuf gene, nox gene and ms53_0285 gene were used for PCR reaction. The primers and PCR reaction conditions are shown in Table 2 below (primer sequences are shown in Table 1 (Shown in above); 50 μL of the PCR reaction mixture contains 1-times GDP-HiFi PCR buffer B, 200 μM of dATP, dTTP, dGTP, and dCTP, 1 μM amplification primer, 200 ng poultry synovial mold and 1 U GDP-HiFi DNA polymerase. After the PCR reaction, agarose gel electrophoresis was used to confirm the presence or absence of DNA fragments of the estimated size.

Table 2: PCR reaction conditions and primer pairs.

3. Recovery and selection of PCR products.

The PCR Clean Up system kit (GMbiolab, Taiwan) was used to recover the PCR products according to the product instructions. The CloneJET PCR Cloning Kit was then used to select the agarase gene. The experimental procedure of breeding is performed with reference to the operation manual provided by the manufacturer. The adhesion mixture was transformed into E. coli ECOS ^™ 9-5. The detailed steps of transformation can refer to the product instructions, or can be modified and adjusted by standard experimental procedures in the field.

The transformed cells were cultured on LB solid medium containing ampicillin (100 μg / mL). After the growth of the bacteria is delayed, the colony polymerase chain reaction (colony PCR) is used to screen the transformants. First, configure 100 μL of PCR reaction solution, including 1x Taq reaction buffer, 200 μM dNTP (dATP, dTTP, dGTP, and dCTP), 1 μM amplification primer, and 2.5 U DreamTaq DNA polymerase (Thermo, USA). Aliquot the PCR reaction solution into a PCR vial (10 μL / tube). After using the toothpick to point the colonies to the PCR vial, the PCR reaction can be performed. PCR reaction conditions are: 5 minutes reaction at 95 ° C; 30 seconds reaction at 95 ° C, 30 seconds reaction at 55 ° C, 72 minutes at 55 ° C, and X minutes (25 cycles); 72 minutes reaction at 72 ° C: X minutes is the DNA polymerase extension time The time set for the tuf gene and the nox gene is 1 minute and 30 seconds, and the time set for the ms53_0285 gene is 2 minutes and 30 seconds. After the PCR reaction was completed, the results of PCR were observed by agarose gel electrophoresis. Colony PCR was used to confirm that the recombinant plastids in the transformed strains had extraneous DNA (insert DNA), and then the plastids in the transformed strains were extracted and DNA sequenced (Yuanzi International Biotechnology Co., Ltd.). The plastids containing tuf gene, nox gene and ms53_0285 gene were named pJET-TUF, pJET-NOX and pJET-MS53, respectively.

4. Site-directed mutation of nox gene.

The nox gene carries three TGA codons. The TGA codon can be translated to tryptophan in mold mold, but is regarded as a stop codon in E. coli. In order to avoid the inability to use the E. coli expression system to produce complete proteins, a point mutation must be performed on the TGA codon in the nox gene and replaced with a codon that E. coli can recognize and translate to tryptophan (replaced with TGG).

The design principles of mutation primers required for point mutation include that the mutation point is located in the center of the primer and the Tm temperature of the primer needs to be higher than 78 ° C. The Tm value of the mutated primer is calculated by the formula Tm = 81.5 + 0.41 (% GC)-675 / N-% mismatch provided by Invitrogene. The% GC in the formula is the percentage of GC in the primer nucleotides; N is the primer Length;% mismatch is the percentage of the mutated base in the primer nucleotides. The point mutation program for TGA in the nox gene uses the primer pairs described in Table 3 below, including the combination of NOXBAMHIF / NOXM2, NOXM1 / NOXM4, NOXM3 / NOXM6, and NOXM5 / NOXSALIR.

Table 3: Primers used in the point mutation program of the nox gene.

The 50 μL PCR reaction mixture contains 1-times GDP-HiFi PCR buffer B, 200 μM of dATP, dTTP, dGTP, and dCTP, 1 μM amplification primer, 100 ng pJET-NOX, and 1 U GDP-HiFi DNA polymerase. The conditions of the PCR reaction were 96 minutes reaction for 2 minutes; 94 ° C reaction for 30 seconds, 55 ° C reaction for 30 seconds, and 68 ° C reaction for 30 seconds (35 cycles); 68 ° C reaction for 5 minutes.

After the PCR reaction, agarose gel electrophoresis was used to confirm the presence or absence of DNA fragments of the estimated size. The PCR products were recovered using a Gel-M ^™ gel extraction system kit. After that, using the recovered four PCR products as templates, the gene amplification was performed by using the NOXBAMHIF / NOXSALIR primer combination. The conditions of the PCR reaction were 96 minutes reaction for 2 minutes; 94 ° C reaction for 30 seconds, 55 ° C reaction for 30 seconds, 68 ° C reaction for 45 seconds (35 cycles), and 68 ° C reaction for 5 minutes. After this step, the full-length site-directed nox gene can be obtained. The PCR Clean Up kit was used to recover the PCR products.

5. Site -directed mutation of ms53_0285 gene.

The ms53_0285 gene contains six TGA codons. The point mutation program for the TGA in the ms53_0285 gene uses the primer pairs described in Table 4 below, including MS53BAMHIF / MS53M2, MS53M1 / MS53M4, MS53M3 / MS53M6, MS53M5 / MS53M8, MS53M7 / MS53M10, MS53M9 / MS53M12, and MS53M11 / MS53SALIR Of combination.

Table 4: Primers used in the point mutation program of the ms53_0285 gene.

The 50 μL PCR reaction mixture contains 1-times GDP-HiFi PCR buffer B, 200 μM of dATP, dTTP, dGTP and dCTP, 1 μM amplification primer, 100 ng pJET-MS53 and 1 U GDP-HiFi DNA polymerase. The conditions of the PCR reaction were 96 minutes reaction for 2 minutes; 94 ° C reaction for 30 seconds, 55 ° C reaction for 30 seconds, and 68 ° C reaction for 30 seconds (35 cycles); 68 ° C reaction for 5 minutes.

After the PCR reaction, agarose gel electrophoresis was used to confirm the presence or absence of DNA fragments of the estimated size. The PCR products were recovered using a Gel-M ^™ gel extraction system kit. Then, using the recovered seven PCR products as templates, the gene amplification was performed using the MS53BAMHIF / MS53SALIR primer combination. The conditions of the PCR reaction were 96 minutes reaction for 2 minutes; 94 ° C reaction for 30 seconds, 55 ° C reaction for 30 seconds, and 68 ° C reaction for 1 minute and 15 seconds (35 cycles); 68 ° C reaction for 5 minutes. After this step, the full-length site-directed mutation of ms53_0285 gene can be obtained. PCR Clean Up kit was used for PCR product recovery.

After the foregoing operations, the present invention obtains the tuf gene (SEQ ID NO: 04), the nox gene (SEQ ID NO: 05), and the ms53_0285 gene (SEQ ID NO: 06), which can be translated into Tuf (SEQ ID NO), respectively. : 01), Nox (SEQ ID NO: 02), and MS53_028520 (SEQ ID NO: 03) proteins.

Experiment three: The construction of the poultry synovial mold antigen expressing plastid of the present invention.

The plastids containing the dsbC gene of E. coli were used as the backbone to construct the plastids of the poultry synovial mold. The construction process of performance plastid is as follows.

1. Tuf represents the construction of plastids.

After the amplified tuf gene was cut with Bam HI and Sal I, the DNA fragment was inserted into the DsbC fusion expression plastid pET-HISDsbC with the same restriction enzyme using T4 DNA ligase. The adhesion product was transformed into E. coli ECOS 9-5. The colony polymerase chain reaction was used to screen the transformants. Use DNA electrophoresis to confirm the presence of DNA fragments of the estimated size. After confirming that the recombinant plastids in the transformed strains have extraneous DNA, the plastids in the transformed strains were extracted and sequenced. The plastids with correct DNA sequences were named pET-HISDsbC-MSTUF (SEQ ID NO: 07).

2. Nox represents the construction of plastids.

After cutting the mutated nox gene with Bam HI and Sal I, the DNA fragment was inserted into the ps-DsbC ps-DsbC fusion by DsbC fusion with the same restriction enzyme using T4 DNA ligase. The adhesion product was transformed into E. coli ECOS 9-5. The colony polymerase chain reaction was used to screen the transformants. Use DNA electrophoresis to confirm the presence of DNA fragments of the estimated size. After confirming that the recombinant plastids in the transformed strains have extraneous DNA, the plastids in the transformed strains were extracted and sequenced. The plastids with correct DNA sequences were named pET-DsbC-MSNOX (SEQ ID NO: 08).

3. MS53_0285 represents the construction of plastids.

After the mutant ms53_0285 gene was cleaved with Bam HI and Sal I, the DNA fragment was inserted into the DsbC fusion expression plastid pET-DsbC with the same restriction enzyme using T4 DNA ligase. The adhesion product was transformed into E. coli ECOS 9-5. The colony polymerase chain reaction was used to screen the transformants. Use DNA electrophoresis to confirm the presence of DNA fragments of the estimated size. After confirming that the recombinant plastids in the transformed strains have extraneous DNA, the plastids in the transformed strains were extracted and sequenced. The plastids with correct DNA sequences were named pET-DsbC-MS53 (SEQ ID NO: 09).

Experiment 4: Expression and purification of recombinant poultry synovial mold antigen.

The antigen-presenting plastids were transformed into E. coli BL21 (DE3). A single colony of the expressing strain was selected and inoculated in 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL), and cultured overnight at 37 ° C and 180 rpm. Then, take 10 mL of this culture bacterial solution and add it to 1 L of LB medium containing kanamycin (final concentration: 30 μg / mL), and shake culture (37 ° C, 180 rpm) to an OD _{600 of} about 0.4 ~ 0.6 about. Then, the expression was induced by adding 0.1 mM isopropyl-β-D-thiogalactoside at 28 ° C. After 4 hours of induction, the cells were collected by centrifugation (10,000 × g, 10 minutes, 4 ° C). The cells were then suspended in 10 mL of a phosphate buffer solution (20 mM sodium phosphate, 500 mM NaCl, pH 7.4), and the cells were broken with an ultrasonic breaker, and then centrifuged (30,966 × g, 30 minutes) to Collect the supernatant fraction. Finally, the collected supernatant was filtered through a 0.22 µm filter membrane. The expression and solubility of the recombinant antigen were observed by protein electrophoresis.

Then, the protein was purified by immobilized-metal ion affinity chromatography using the characteristic that the recombinant antigen with a His tag can form a covalent covalent bond with nickel or cobalt ions. The recombinant antigen was purified using a protein liquid chromatography system ÄKTA prime plus (GE Healthcare, Sweden) with a 5 mL HiTrap ^™ Ni excel column (GE Healthcare, Sweden). First, after equilibrating the column with 25 mL of a phosphate buffer solution, the aforementioned supernatant was injected into a HiTrap ^™ Ni excel column. After the injection of the sample is completed, wash with 100 mL of 30 mM imidazole washing buffer (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4) unless the protein is specifically bound. Finally, the recombinant antigen on the resin was extracted with 150 mL of a dissociation buffer containing 250 mM imidazole (20 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4). The principle is to compete with the recombinant antigen for the resin with a high concentration of imidazole The binding site caused the recombinant antigen to be eluted from the resin. The purified antigen solution was placed in an Amicon ^™ ultra-15 ultracel-30K centrifuge tube (Merck Millipore, USA), centrifuged at 2,600 × g to an appropriate volume at 4 ° C, and stored at 4 ° C for later use.

The experimental results are shown in the first figure. The first graph A shows that the antigens shown in this experiment have excellent solubility, which is conducive to the subsequent purification process and commercial implementation. The first panel B shows that the purified antigen has a reliable purity.

Experiment five: The preparation of the single antigen vaccine of the present invention and chicken tests.

Obtain 4 week-old chickens without specific pathogens (purchased from the Animal Medicine Testing Branch of the Animal Health Test Institute of the Agricultural Commission of the Executive Yuan) and keep them in the certified animal houses of the Institute. Recombinant poultry synovial mycoplasma antigens Tuf, NOX and MS53_0285 were mixed with the commercially available adjuvant MONTANIDE ™ Gel 01 (Seppic) according to the product instructions, so that the final dose of each single antigen vaccine was 50 μg / 0.5 mL. At 4 weeks of age, the chickens were injected with 0.5 mL of the above-mentioned single-antigen vaccine subcutaneously in the back of the neck, and then immunized for a second time at 6 weeks of age. After the immunization, they were observed for two weeks and blood was collected. × g, 4 ° C, 15 minutes). Serum was collected, and a commercially available kit (IDEXX Mycoplasma synoviae Antibody Test Kit) was used to measure poultry synovia mold antibody.

The experimental results are shown in Table 5 and the second figure below. The average serum antibodies of chickens administered with the Tuf, NOX, and MS53_0285 single antigen vaccines of the present invention all showed positive reactions, and the positive rates reached 57%, 71%, and 75%, respectively.

Table 5: Records of antibody positive rates for single antigen vaccines.

Then, at the age of 8 weeks, the chickens were challenged with tracheal and foot pads using a synovial mold of poultry. The attack strain used was ATRI 2014 (hereinafter referred to as MS-f1), which was isolated from a commercial chicken farm in Taiwan. Of the strain. The strain was inoculated in a mold culture medium and cultured at 37 ° C and 75 rpm for 32 hours.

For the tracheal infection group, place the chicken in Baoding, then insert the feeding trachea into the trachea of the chicken, and then use a syringe to inject 1 mL of bacteriostatic solution containing 10 ⁶ CCU bacteria. The liquid spreads evenly. For groups infected with foot pads, first wipe the chicken foot pads with alcohol cotton, use the needle to insert the chicken foot pads, and then inject 0.5 ml of bacteriostatic liquid containing 10 ³ CCU bacteria. In addition to collecting blood samples and separating serum to evaluate the specific antibodies to synovial mold of poultry after the challenge, the body weight, clinical foot pad temperature and thickness of the chickens at each time point were also recorded, and the chickens were dissected and observed after the test. Symptoms such as trachea, balloon, and footpad swelling are recorded according to the evaluation index of organ lesions. The lesion scoring criteria are shown in Table 6 below.

Table 6: Scoring criteria for poultry anatomy lesions.

When observing the thickness of the foot pads of chickens, it was found that the degree of swelling of the foot pads of the chickens administered with the single antigen vaccine of the present invention was slightly less than that of the untreated group (Figure 3). On the other hand, measuring the temperature of chicken foot pads showed that the single-antigen vaccine of the present invention improved the degree of inflammation of chicken foot pads (Figure 4). Further evaluation and recording of the footpad lesion score showed that chickens administered the single-antigen vaccine of the present invention had fewer footpad lesions, of which Tuf vaccine was particularly effective (figure 5).

Airbag lesions are also typical of poultry synovial mold infections. The air sacs of normal chickens should be clear and transparent with no secretions. The sixth figure A shows that the airbags of chickens to which the single-antigen vaccine of the present invention has not been administered are significantly thickened and yellow cheese-like fibrin is attached. In contrast, administration of the single-antigen vaccine of the present invention can effectively prevent airbag lesions, and the experimental results show that the effects of the NOX vaccine and the MS53_0285 vaccine of the present invention are particularly significant.

The trachea of the chicken was further sectioned and tissue stained (H & E staining) to observe the change of tracheal ring mucosa thickness. The results show that the single antigen vaccine of the present invention can effectively prevent tracheal mucosal lesions compared with chickens not administered the single antigen vaccine of the present invention, and the effect of the subunit vaccine of MS53_0285 is the best (sixth figure B).

According to the results of the previous disclosure, although the Tuf vaccine, the NOX vaccine and the MS53_0285 vaccine of the present invention have slightly different effects, they can alleviate the symptoms caused by the synovial mold of poultry.

Experiment 6: Preparation and chicken test of the multi-antigen vaccine (cocktail vaccine) of the present invention .

Obtain 4 week-old chickens without specific pathogens (purchased from the Animal Medicine Testing Branch of the Animal Health Test Institute of the Agricultural Commission of the Executive Yuan) and keep them in the certified animal houses of the Institute. A number of cocktail vaccines of the present invention were prepared by mixing commercially available adjuvants with different antigen combinations (Tuf + NOX, Tuf + MS53_0285, NOX + MS53_0285, and Tuf + NOX + MS53_0285). Each vaccine (0.5 mL) contains 50 μg of each antigen.

When the chickens were 4 weeks of age, 0.5 mL of the above combination antigen vaccine was injected subcutaneously in the back of the neck, and a second immunization was performed at 6 weeks of age. After the immunization, the chickens were recorded for two weeks and blood was collected, and then centrifuged (2,000 × g, 4 ° C, 15 minutes) to collect serum and detect specific antibodies using a commercially available kit (IDEXX Mycoplasma synoviae Antibody Test Kit).

The experimental results are shown in Tables 7 and 7 below. The results showed that both the double antigen vaccine and the triple antigen vaccine enhanced the antibody performance in chickens. In other words, the recombinant antigen of the present invention not only does not have the disadvantage of mutual interference when used in combination, but has the potential of synergistic effect to improve the immune-inducing effect.

Table seven:

Experiment 7: Preparation and chicken test of the multi-antigen vaccine (cocktail vaccine) of the present invention.

Obtain 4 week-old chickens without specific pathogens (purchased from the Animal Medicine Testing Branch of the Animal Health Test Institute of the Agricultural Commission of the Executive Yuan) and keep them in the certified animal houses of the Institute. The cocktail antigen of the present invention is prepared by mixing the recombinant antigen of the present invention, Tuf, NOX and MS53_0285 with a commercially available adjuvant MONTANIDE ™ ISA 71 VG (Seppic). Each vaccine (0.5 mL) contains 50 μg of each antigen. The adjuvant and the antigen are adjusted at a weight ratio of 7: 3. The specific gravity of the aforementioned adjuvant is about 0.816, so about 0.37 mL of the vaccine prepared in this experiment is an adjuvant.

When the chickens were 4 weeks of age, 0.5 mL of the above combination antigen vaccine was injected subcutaneously in the back of the neck, and a second immunization was performed at 6 weeks of age. After the immunization, the chickens were recorded for two weeks and blood was collected, and then centrifuged (2,000 × g, 4 ° C, 15 minutes) to collect serum and detect specific antibodies using a commercially available kit (IDEXX Mycoplasma synoviae Antibody Test Kit).

The experimental results are shown in Table 8 and Figure 8 below. The results showed that the tri-antigen vaccine induced antibody expression in chickens. In other words, the recombinant antigen of the present invention not only does not have the disadvantage of mutual interference when used in combination, but has the potential of synergistic effect to improve the immune-inducing effect. The results of this experiment are consistent with those shown in the seventh figure of the previous publication.

Table eight:

Then, at the age of 8 weeks, chickens were challenged with tracheal and footpads with poultry synovial mold MS-f1. After the challenge, in addition to collecting blood samples to isolate serum to evaluate the specific antibodies against poultry synovial mold, The body weight, temperature and thickness of the foot pads were also recorded at each time point. After the test, the animals were dissected and the swelling symptoms of the air sacs and foot pads were observed.

Experimental results show that the tri-antigen vaccine of the present invention can reduce the weight loss of chickens after infection (Figure 9). In addition, the tri-antigen vaccine of the present invention also showed significant effects on the symptoms of foot pad swelling and foot pad temperature (Figures 10 and 11). After further sacrificing the chickens and observing the results of the footpad and airbag lesion scoring results, it was found that the tri-antigen vaccine of the present invention has a tendency to reduce footpad and airbag lesions.

Table 9:

Experiment 8: Dose test of the tri-antigen vaccine of the present invention .

The recombinant antigen of the present invention is mixed with a commercially available adjuvant MONTANIDE ™ ISA 71 VG (Seppic), and each dose (0.5 mL) contains 150 μg, 100 μg, 50 μg, 20 μg and other four different concentrations of the recombinant antigen. For a unit vaccine, the adjuvant and the antigen are adjusted at a weight ratio of 7: 3. The specific gravity of the aforementioned adjuvant is about 0.816, so about 0.37 mL of the vaccine prepared in this experiment is an adjuvant. The group design is shown in Table 10.

Table ten:

Obtain 4 week-old chickens without specific pathogens (purchased from the Animal Medicine Testing Branch of the Animal Health Test Institute of the Agricultural Commission of the Executive Yuan) and keep them in the certified animal houses of the Institute. The chickens were immunized with the above-mentioned combination antigen vaccine two times at two-week intervals. Then two weeks later, poultry synovial mold MS-f1 was used for trachea and foot pad challenge. After the challenge, in addition to collecting blood samples and separating serum to evaluate the specific antibodies to the synovial mold of poultry, the body weight, foot pad temperature and thickness of the chickens were recorded at various time points. The airbag symptoms were scored according to the evaluation index of organ lesions in Table 5.

The experimental results are shown in Tables 11 and 12 below. The results showed that all four doses of this experiment could induce antibody performance in chickens. In addition, all four doses tested in this experiment were effective in alleviating symptoms such as body weight and foot pad temperature (Figures 13 and 14). The birds were sacrificed to observe the airbag lesions. The results showed that the four doses tested in this experiment were all effective in improving balloon lesions (Table 12).

Table 11:

Table 12:

no

The first figure shows the experimental results of Experiment 4 of the present invention. (A) The solubility of the recombinant antigen produced by the present invention is examined by protein electrophoresis (T represents whole cell lysate, and S represents soluble part). (B) The purity of the purified recombinant antigen of the present invention is examined by protein electrophoresis.

The second figure shows the positive reaction of the poultry synovial mold-specific antibodies in the chicken serum of the experiment 5 of the present invention.

The third figure shows the degree of increase in footpad thickness of chickens in the fifth experimental experiment of the present invention after infection with poultry synovial mold.

The fourth figure shows the degree of increase in foot pad temperature of chickens in the fifth experimental experiment of the present invention after infected with poultry synovial mold.

The fifth graph shows the scores of the footpad lesions of the chickens in the fifth experimental experiment of the present invention after infection with poultry synovial mold.

The sixth figure shows the airbag lesion score (A) of the chickens in the fifth experimental experiment of the present invention after infection with poultry synovial mold, and the thickness of the tracheal mucosa (B) of the fifth experimental chickens. Statistical indicators show that the vaccine of the present invention is statistically significant compared to the positive control group (T-test, †: p> 0.1 ).

The seventh figure shows the positive reaction of the poultry synovial mold-specific antibody in the chicken serum of the experiment 6 of the present invention.

The eighth figure shows the positive reaction of the poultry synovial mold-specific antibody in the chicken serum of the experiment 7 of the present invention.

The ninth figure shows the change in body weight of the chickens of the experiment 7 of the present invention after infection with the synovial mold of poultry.

The tenth figure shows the change in the thickness of the foot pads of the chickens of the experiment seven of the present invention after being infected with the synovial mold of poultry. Statistical indicators show that the vaccine of the present invention is statistically significant compared to the positive control group (T-test, †: p> 0.1 ).

The eleventh figure shows the temperature change of the foot pads of the chickens of the experiment 7 of the present invention after being infected by the synovial mold of poultry. Statistical indicators show that the vaccine of the present invention is statistically significant compared to the positive control group (T-test, *: p> 0.05 ).

The twelfth figure shows the positive reaction of poultry synovial mold-specific antibodies in the chicken sera of Experiment 8 of the present invention.

The thirteenth figure shows the weight change of the chickens of the experiment eight of the present invention after infected with the synovial mold of poultry. Statistical indicators show that the vaccine of the present invention is statistically significant compared to the positive control group (T-test, *: p> 0.05 ).

The fourteenth figure shows the temperature change of the foot pads of the chickens of the experiment eight of the present invention after infected with the synovial mold of poultry. Statistical indicators show that the vaccine of the present invention is statistically significant compared to the positive control group (T-test, †: p>0.1; *: p> 0.05 ).

no

Claims (13)

A composition for reducing the symptoms caused by a synovial mold infection of poultry, comprising: An active ingredient comprising NOX, MS53-0285, or a combination thereof; wherein the NOX comprises the sequence shown in SEQ ID NO: 02, and the aforementioned MS53-0285 comprises the sequence shown in SEQ ID NO: 03; and A pharmaceutically acceptable adjuvant. The composition according to claim 1, wherein the concentration of the aforementioned active ingredient is 40 to 900 μg / mL, which is based on the total volume of the aforementioned composition. The composition according to claim 1, wherein the pharmaceutically acceptable adjuvant is: Freund's complete or incomplete adjuvant, aluminum gel, surfactant, polyanion, peptide, oil emulsion, or a combination thereof. The composition according to claim 1, further comprising a pharmaceutically acceptable additive. The composition according to claim 4, wherein the pharmaceutically acceptable additive is a solvent, a stabilizer, a diluent, a preservative, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent, or a combination thereof. The composition according to claim 1, wherein the above-mentioned symptoms caused by the infection of the poultry synovial mold are tracheal disease, balloon disease, arthritis, or a combination thereof. According to the composition described in claim 1, when the aforementioned active ingredient contains NOX, MS53-0285, or a combination thereof, the aforementioned symptoms caused by the infection of synovial mycoplasma of poultry are at least airbag lesions. According to the composition of claim 1, when the aforementioned active ingredient comprises MS53-0285, the aforementioned symptoms caused by the infection of the synovial mold of the poultry are at least tracheal lesions. A performance vector comprising: A nucleotide sequence having the sequence shown in SEQ ID NO: 05, SEQ ID NO: 06, or a combination thereof; A performance element comprising a promoter and a ribosome binding site; and A fusion partner sequence. A performance vector comprising: A nucleotide sequence translated into a protein of NOX, MS53-0285, or a combination thereof; wherein the NOX comprises the sequence shown in SEQ ID NO: 02, and the aforementioned MS53-0285 comprises the sequence shown in SEQ ID NO: 03; A performance element comprising a promoter and a ribosome binding site; and A fusion partner sequence. The expression vector according to claim 9 or 10, wherein the fusion partner is DsbC of E. coli, MsyB of E. coli, FklB of E. coli, or a combination thereof. The expression vector according to claim 9 or 10, which comprises the sequence shown in SEQ ID NO: 08 or EQ ID NO: 09. Use of a protein for preparing a composition for reducing symptoms caused by a synovial mycoplasma infection of poultry, wherein the aforementioned protein comprises NOX, MS53-0285, or a combination thereof; wherein the aforementioned NOX has the sequence shown in SEQ ID NO: 02 The aforementioned MS53-0285 has the sequence shown in SEQ ID NO: 03.