TWI614026B - Recombinant avibacterium paragallinarum flfa fimbrium subunit vaccine for infectious coryza of chickens, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a recombinant FlfA pilin protein using Avibacterium paragallinarum as a secondary unit vaccine. The invention further provides a method for preparing the recombinant FlfA pilin vaccine, comprising the method for producing a recombinant FlfA pilin vaccine by Escherichia coli, and using the obtained recombinant protein as an effective vaccine composition for protecting chicken against infectious insects A method of infection with rhinitis. According to the results of animal experiments, the sub-unit vaccine of the present invention can produce a good protective effect after the application of the chicken, and can combat the infectious nephritis infection of chickens of 10 ⁷ -10 ⁹ CFU.

Description

Recombinant FlfA pilin subunit vaccine of chicken infectious rhinitis and preparation and application thereof law

The present invention relates to an infectious coryza vaccine, particularly with regard to infectious coryza comprising (Avibacterium paragallinarum) strain of recombinant FlfA fiber protein subunit vaccines.

Infectious coryza (IC), also known as chicken infectious rhinitis, is a chicken respiratory infection caused by Avibacterium paragallinarumm ( Av . paragallinarumm ). Infectious rhinitis is classified as a Gram-negative bacterium belonging to the family Pasteurellaceae (Blackall et al., Int J Syst Evol Microbiol 55 (1): 353-62, 2005), and the chicken is infected. It can lead to problems such as growth retardation and decreased egg production rate, and cause serious economic losses. Infectious measurables must be controlled by vaccines. The number of infectious vaccination vaccines used every year around the world is also very large. The currently commercial chicken infectious rhinitis vaccines are all cultivating chicken infectious rhinobacteria in liquid medium. The whole fungus of Av.paragallinarum is produced.

While conventional whole-bacterial inactivated vaccines provide chicken protection, such vaccines have a number of disadvantages. First, chicken infectious rhinitis bacteria are known to be classified into three serotypes A, B and C (Blackall et al., Avian Dis 34 (3): 643-5, 1990), while the protection of conventional whole-bacterial inactivated vaccines is known. Limited to strains of the same serotype, lacking cross-protection between different serotypes (Blackall & Soriano: Edited by Swayne et al., Diseases of poultry , 13th edition, published by John Wiley & Sons. Inc.; p.859- 873, 2013).

Second, the in vitro culture of most chicken infectious rhinitis bacteria requires a medium containing nicotinamide adenine dinucleotide (NAD) (Rimler, RB et al., 1977. J Gen Microbiol 98 (2): 349 -54), and some Av. paragallinarum strains require additional chicken serum (Blackall & Reid, Vet Microbiol 7 (4): 359-67, 1982.), thus preparing the medium required for the conventional whole-plasma inactivated vaccine. Expensive and time consuming, the production cost of the vaccine is high.

Third, since the preparation of the conventional whole-bacteria inactivated vaccine requires emulsification with an oil adjuvant, the chicken may have local allergic reactions and granuloma after the whole vaccine is immunized. Therefore, how to develop a new generation of anti-chicken infectious rhinitis vaccine with low price, low side effects and cross-protection of different serotypes is the target of many chicken infectious rhinitis vaccine developers around the world.

Previous studies have found that many of the pathogens of the genus Pasteurella have a structure of fimbriae, which is involved in the adsorption of bacteria on the surface of host cells (Jacques & Paradis, FEMS Microbiol Rev 22 :45-59, 1998). Many studies have pointed out that cilia are one of the important virulence factors of bacteria (Klemm & Schembri, Int J Med Microbiol 290 : 27-35, 2000). In recent years, some scholars have found that Gallibacterium anatis ( G.anatis ), which is closely related to chicken infectious rhinitis, also has a cilia structure, and the cilia gene sequence found in the bacterium is a so-called "F17" family cilia (Bager et al., Infect Immun 81 : 1964-73, 2013).

et al.,BMC Genomics 15：1093,2014)，但在這些已知的卡氏桿菌FlfA纖毛蛋白中，只有來自Gallibacterium anatis 12656-12菌株之FlfA纖毛蛋白，被證明能有效作為次單位疫苗(Bager et al.,Infect Immun 81,1964-73,2013)。 "F17" family cilia of the biosynthetic gene is comprised of four genes (flfACDG) consisting of (Dobrindt et al, Infect Immun 70 :. 6365-6372,2002), which went flfA the translation of the gene protein as the main FlfA Cilia structural protein. Research in the card's bacillus (G.anatis) the discovery, recombinant protein FlfA performance of E.coli can be used as subunit vaccines to stimulate the immune system to produce antibodies against the card's valid bacillus of attack (Bager et al., Infect Immun 81 , 1964-73, 2013). Although at least 15 sequences of FlfA pilin from Gallibacterium spp. have been published (Kudirkien

Et al., BMC Genomics 15 :1093, 2014), but among these known K. faecalis FlfA pilin proteins, only FlfA pilin from Gallibacterium anatis 12656-12 strain has been shown to be effective as a secondary unit vaccine (Bager Et al., Infect Immun 81 , 1964-73, 2013).

Thus, the present invention has been developed to prepare a vaccine for the prevention and treatment of infectious diseases of chicken infectious rhinitis by using FGFA pilin protein of chicken infectious rhinitis.

The present invention is based on the above object, and it has been found by whole genome sequencing and annotation techniques that Av. paragallinarum has the FlfA gene and also has the ability to express FlfA pilin. On the other hand, recombinant FlfA pilin protein produced by E. coli can achieve a satisfactory yield and can be used as an effective sub-unit vaccine to protect chickens against infectious rhinovirus infections.

Thus, in one aspect, the invention relates to a subunit vaccine composition for controlling an animal disease caused by avian infectious rhinobacteria Av . paragallinarum , characterized by comprising a FlfA of Avibacterium paragallinarum The pilin protein or fragment thereof is used as an antigen and a veterinary acceptable adjuvant.

In some embodiments of the invention, the F. avian infectious rhinitis FlfA pilin protein is selected from the group consisting of amino acid sequences listed in any one of SEQ ID No. 1 to SEQ ID No. 7 of the Sequence Listing. A protein of more than 95%, preferably more than 95% similar amino acid sequence. In other specific aspects, the chicken infectious rhinobacteria FlfA pilin protein has a protein as selected from the amino acid sequences set forth in any one of SEQ ID No. 1 to SEQ ID No. 7 of the Sequence Listing.

In other specific aspects of the invention, the animal disease is infectious coryza. In other specific aspects of the invention, the animal is a chicken, especially a breeder, laying hen, or colored chicken.

Figure 1 (A) shows the structure of the flfADCG gene of Av . paragallinarum , and the position map of FlfA (+) and FlfG (-) primer pairs. The dotted line is where the PCR product is located. Fig. 1(B) is an electrophoresis analysis diagram of PCR reaction using FlfA(+) and FlfG(-) primer pairs. The amplified product of 5.1 kb can be amplified from all strains of Av. paragallinarum isolated from Taiwan. The flfADCG genome is ubiquitous in Taiwan's Av. paragallinarum strain.

Figure 2 (A) shows the results of analyzing the crude extract and purified product of the r-FlfA recombinant protein expressed by E. coli by SDS-polyacrylamide electrophoresis (SDS-PAGE). Lane M is a molecular weight marker. Figure 2 (B) shows the results of Western blot analysis of a single antibody against anti-hexa-histidine, and the position of the recombinant r-FlfA protein (20 kDa) (indicated by an arrow) can be confirmed.

The specific terms used in the present invention have their original meanings, and certain specific terms are used as follows to provide those skilled in the art to further understand the present invention. For convenience, some special terms will be indicated in italics or quotation marks, but these marked parts will not affect the scope or meaning of the special term itself, just like the unmarked text in this article. That is to say, the same thing will have more than one statement.

The terms "infectious susceptibility (IC)" and "chicken infectious rhinitis" as used herein are used interchangeably in the specification, meaning that the chicken is caused by avian parasitic bacterium ( Av. paragallinarum ). Respiratory infectious diseases.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example

Example 1: Expression and analysis of recombinant FlfA pilin protein of avian infectious rhinitis Av. paragallinarum

To explore whether the bacteria infectious coryza performance capabilities of the fiber protein of the present invention, firstly the whole bacterial genome sequencing and annotation technology found in strains isolated TW07 Av.paragallinarum Taiwan, there is the card's bacillus (G.anatis) A similar cilia genome, flfADCG (Fig. 1A) (Liu et al., 2016, Avian Dis , 2016, in press). A specific primer pair was designed based on this flfADCG sequence, namely FlfA(+): 5'-ATG AAA AAA TTG GTT TTA ACA-3' (SEQ ID NO. 8), and FlfG(-): 5'-TCA TAA TAA ATA TTG TAA GTC-3' (SEQ ID NO. 9) (Fig. 1A) was amplified by PCR, and a DNA fragment of about 5.1 kb in length was amplified from all strains of Av. paragallinarum isolated from Taiwan (Fig. 1B). Nucleic acid sequencing is then performed. The sequencing method was performed using a sequencer (ABI-3730XL DNA Analyzer, Applied Biosystem) to perform nucleic acid sequence confirmation in a sequencing kit BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The sequencing results revealed that these 5.1 kb DNA fragments contained the complete flfADCG gene, confirming that the flfADCG gene is ubiquitous in the isolated Av . paragallinarum in Taiwan.

Subsequently, a total of seven bacterial infectious coryza (Av.paragallinarum TW07, TW09a, TW11, TW12-2, TW12-3, TW13, TW14 and isolate) based on the case of the registered person Labs invention flfADC G gene sequence (Accession Number=KU757292-KU757298), PCR primer pair was designed to increase the flfA gene. The primer pair used was r-FlfA(+): 5'-CCA TGG ACC AAA CAA ACT CAG GAA CT-3' (SEQ ID NO. 10) and r-FlfA (-): 5'-GTC GAC TTC GTA TGC GAT GGT ATA ATT-3' (SEQ ID NO. 11), the 5' end of the above primer sequence, has an enzyme cleavage site for restriction enzymes Nco I and Sal I, respectively, for colonization into expression vectors. First, the flf A gene was amplified by PCR reaction, and then the amplified product was designed to bind to the enzyme, then ligated into the expression vector pET28a (Novagen, Inc. Madison, WI, USA), and finally confirmed the plastid sequence in sequence, which was correct. The performance of the recombinant r-FlfA protein was then performed. The amino acid sequences of the flf A proteins of the seven Taiwan field isolates analyzed in Taiwan were as shown in SEQ ID No. 1-7, respectively, and their similarities were 100%.

On the other hand, the results of the sequence alignment shown below show that the amino acid sequence of the previously known FlfA protein of Gallibacterium anatis 12656-12 and the amino acid sequence of the FlfA protein of Av . paragallinarum of the present invention, The similarity is only 71%. It may be due to the different functions of the two, or because Av. paragallinarum and Gallibacterium anatis are classified into different species and different genera, and the diseases caused by the infected animals are also different.

Performance of recombinant r-FlfA protein

The constructed expression vector was transformed into E. coli BL21 (DE3) host cells, and cultured in LB medium at 37 ° C until the absorbance (OD ₆₀₀ ) reached 0.6, and IPTG (isopropyl thio) was added. -β-D-galactoside) The final concentration was 0.4 mM, and transcription of the induced gene was carried out to synthesize a large amount of recombinant r-FlfA expression protein. The E. coli cells expressing the recombinant protein were then suspended in a binding buffer (20 mM pH 7.9 Tris, 5 mM imidazole, 500 mM NaCl) and disrupted by ultrasonic waves. Thereafter, the mixture was centrifuged at 12,000 rpm for 30 minutes at 4 ° C, and the supernatant containing the soluble protein was collected, and the recombinant protein was purified by using a column containing 2.5 mL of "His-bind" resin (Novagen) affinity resin.

Purification of recombinant r-FlfA protein

The purification step is as follows. The protein not bound to the affinity resin was first removed with 25 mL of binding buffer and 15 mL of washing buffer (20 mM pH 7.9 Tris, 50 mM imidazole, 500 mM NaCl), and finally 15 mL of the elution buffer was used. The r-FlfA expression protein was recovered (eluting buffer) (20 mM pH 7.9 Tris, 250 mM imidazole, 500 mM NaCl). The purified recombinant protein was assayed in a "Protein Assay" kit (BIO-RAD, Hercules, CA) and subjected to SDS-PAGE and Western blot analysis to observe the expression of recombinant protein and the purity of the protein.

The experimental results are shown in Fig. 2. From the results of Fig. 2A, it was found that the r-FlfA recombinant protein can be expressed in a large amount in E. coli , and the molecular weight of the expressed recombinant protein is about 20 kDa, which is in agreement with the expected molecular weight. Moreover, the r-FlfA recombinant protein can be purified into a single band protein using a column of "His-bind" resin affinity resin. From the results of FIG. 2B, it was found that the anti-His-tag single-source antibody was used for Western blot analysis, and it was found that the r-FlfA protein was recognized by the single-source antibody, and it was revealed that these recombinant proteins were all His-tag-containing fusion proteins. This result is in line with expectations.

Example 2: Preparation of a vaccine against chicken Av. paragallinarum challenge using recombinant r-FlfA protein as antigen

Vaccine efficacy experiments were performed using specific pathogen free (SPF) chickens purchased from the COA's Home Health Center. These SPF chickens were housed in a positive pressure incubation room that was air filtered and periodically before the experiment. Serological tests were performed to determine that they were not infected with Av . paragallinarum .

Preparation of recombinant subunit vaccines is r-FlfA proteins as vaccine antigens, 50 μ g per bird-administration of recombinant proteins, vaccine antigens are 30% emulsion were mixed together with a 70% ratio of the adjuvant ISA70, a control group It was prepared by mixing 30% of PBS buffer and 70% of ISA. The total volume of the vaccine emulsion per chicken was 0.5 mL.

Example 3: Chicken Infectious Rhinitis Av . Paragallinarum Recombinant r-FlfA Protein Subunit Vaccine Chicken Protection

A total of 24 chickens were used in this experiment. Immunization was started at 4 weeks of age, and immunization was repeated two weeks later. 14 days after the second immunization, the wild virulent strain TW07 was used as a challenge strain for needle injection. 150 μL of bacterial fluid was placed in the sinus cavity of the orbital cavity. The dose was 10 ⁹ , 10 ⁸ , and 10 ⁷ colony forming units (CFU), and four chickens were used for each challenge dose. Seven days after challenge, the lesion index of each chicken was assessed daily according to the Bragg Clinical Symptom Assessment Method (Bragg et al., Onderstepoort J Vet Res 69 : 163-169, 2002). After the observation, the chickens were euthanized and the bacteria were detected from the lower sinus of the orbit.

The results after the challenge were as shown in Table 1. When the dose of the challenge was divided into 10 ⁹ , 10 ⁸ , and 10 ⁷ bacteria, the lesion index of the chicken immunized with r-FlfA recombinant protein was 1.32, 0.64, and 0.96. There was a significant decrease (P < 0.01 or P < 0.001) compared to the index of the chickens in the control group that did not receive immunization (2.17, 2.71, 1.42). Therefore, immunization of chickens with the r-FlfA recombinant protein of the present invention does produce the ability to protect chickens vaccinated against Av . paragallinarum .

Based on the experimental results of the above examples, the present invention discloses for the first time that avian infectious rhinitis bacteria can express a pilin protein called FlfA, and can successfully perform a large amount of expression and production of recombinant FlfA protein in E. coli expression system, and can be used as an antigen. A vaccine effective against chicken infectious rhinitis is prepared. Since the medium cost of Escherichia coli is low, and generally, the side effect of the recombinant protein vaccine is lower than that of the whole vaccine, the recombinant FlfA protein subunit vaccine developed by the present technology has excellent market potential.

<110> National Chung Hsing University

<120> Chicken infectious rhinitis recombinant FlfA cilia protein subunit vaccine and preparation and application method thereof

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 194

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW07 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 2

<211> 194

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW09a strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 3

<211>

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW11 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 4

<211> 194

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW012-2 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 5

<211> 194

<212> PRT

<213> Chicken Infectious Rhinitis Av.paragallinarum TW12-3 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 6

<211> 194

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW13 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 7

<211> 194

<212> PRT

<213> Chicken infectious rhinitis strain Av.paragallinarum TW14 strain (serotype C Taiwan field isolate)

<220>

<400>

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 8

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 9

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 10

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400> 11

Claims (4)

A use of a FlfA pilin protein of Avibacterium paragallinarum or a fragment thereof for the preparation of a subunit vaccine for controlling an animal disease caused by avian infectious rhinobacteria Av . paragallinarum , wherein the chicken infectious rhinitis FlfA The pilin protein has a protein as selected from the amino acid sequences listed in any one of SEQ ID No. 1 to SEQ ID No. 7 of the Sequence Listing. The use of claim 1, wherein the animal disease is infectious coryza. The use of claim 1 wherein the animal is a bird. The use of claim 1, wherein the animal is selected from the group consisting of chicken, duck or goose.