# Patent

## Title
Anti-mycoplasma spp. subunit vaccine

## Abstract

Provided in the present invention are anti-Mycoplasma spp. subunit vaccines, especially proteins suitable for being used as the active ingredient of the Mycoplasma spp. subunit vaccines, and a vaccine prepared therefrom. Upon experimenting, it is confirmed that the proteins can elicit an immune response having sufficient strength to avoid the infection of Mycoplasma spp. in pigs. The vaccine can comprise one of the aforementioned proteins as an active ingredient, or can comprise two or more of the proteins to form a form of cocktail vaccine. The vaccine of the present invention is not only more safe than conventional vaccines, but also has equivalent or even better immune effects.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Divisional of application Ser. No. 15/383,962, filed on Dec. 19, 2016, which is a Divisional of application Ser. No. 14/765,512, filed on Aug. 3, 2015 (now U.S. Pat. No. 9,561,267, issued Feb. 7, 2017), which was filed as PCT International Application No. PCT/CN2013/071379 on Feb. 5, 2013, all of which are hereby expressly incorporated by reference into the present application.


FIELD OF THE INVENTION
The present disclosure relates to a vaccine against Mycoplasma spp.; especially to a subunit vaccine against Mycoplasma spp.
BACKGROUND OF THE INVENTION
Mycoplasma spp. is currently known the tiniest bacteria capable of self-replication outside host cells. Although swine enzootic pneumonia would not cause swine death, it will reduce feeding efficiency and cause growth retardation, inflammation, and immunosuppression as well as make swine more vulnerable to infection of other pathogens, which therefore become economic damage of the industry.
So far, swine enzootic pneumonia is prevented by three major strategies, including: medicine administration, environment management, and vaccination. Seeing the bad prevention efficiency of antibiotics to Mycoplasma hyopneumoniae, medicine administration can only used for treatment purposes and is hard to meet prevention needs. Furthermore, considering that drug abuse may lead to a larger infection causing by drug-resistant bacteria, medicine administration needs cautious plans and exists a lot of limitations.
Environment management forms the basis of prevention of Mycoplasma spp. infection. Good piggery sanitation and management would be helpful to reduce occurrence of infection. On the other hand, prevention could be more comprehensive through vaccination.
The conventional vaccines in the field use inactive/dead bacteria as the active ingredient thereof. However, the price of the conventional vaccines is too high because Mycoplasma spp. is fastidious bacteria and is difficult to be cultured in the laboratory. In order to reduce the cost of Mycoplasma spp. vaccines, scientists continuously try to develop vaccines of different types, such as: (1) attenuated vaccines, (2) vector vaccines, (3) subunit vaccines, and (4) DNA vaccines. Among them, subunit vaccines show the most potential because the advantages of ease in production and high safety.
To date, there are several potential candidate proteins that could be used for M. hyopneumoniae vaccines; however, there is no further report verifying the proteins suitable for M. hyopneumoniae vaccines.
SUMMARY OF THE INVENTION
In light of the foregoing, one of the objects of the present invention is to provide antigens suitable for being used in M. hyopneumoniae vaccines and thereby producing novel M. hyopneumoniae vaccines so that the cost of prevention can be reduced.
Another object of the present invention is to provide a combination of antigens that suitable for being used in M. hyopneumoniae vaccines and thereby provide subunit vaccines with better performance; therefore, there would be more options for prevention tasks.
In order to achieve the aforesaid objects, the present invention provides a recombination protein for preparing a vaccine for preventing Mycoplasma spp. infection, comprising an amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a combination thereof.
The present invention also provides a vaccine for preventing Mycoplasma spp. infection, comprising: an active ingredient, comprising a protein of PdhA, XylF, EutD, Mhp145, P78, P132, Mhp389, or a combination thereof; and a pharmaceutically acceptable adjuvant.
Preferably, said active ingredient is of a concentration of 50 to 3500 μg/mL based on the total volume of said vaccine.
Preferably, said active ingredient comprises at least two proteins selected from a group consisting of PdhA, XylF, EutD, Mhp145, P78, P132, and Mhp389.
Preferably, said active ingredient comprises PdhA and P78.
Preferably, said active ingredient comprises XylF and Mhp145.
Preferably, said pharmaceutically acceptable adjuvant is a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.
Preferably, said vaccine further comprises a pharmaceutically acceptable additive.
Preferably, said 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 present invention further provides a vaccine for preventing Mycoplasma spp. infection, comprising: an active ingredient, comprising an amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a combination thereof; and a pharmaceutically acceptable adjuvant.
Preferably, said active ingredient is of a concentration of 50 to 3500 μg/mL based on the total volume of said vaccine.
Preferably, said active ingredient comprises at least two amino acid sequences selected from a group consisting of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
Preferably, said active ingredient comprises amino acid sequences of SEQ ID NO: 08 and SEQ ID NO: 12.
Preferably, said active ingredient comprises amino acid sequences of SEQ ID NO: 09 and SEQ ID NO: 11.
Preferably, said pharmaceutically acceptable adjuvant is a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.
Preferably, said vaccine further comprises a pharmaceutically acceptable additive.
Preferably, said 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 present invention more provides an expression vector for preventing Mycoplasma spp. infection, comprising: a plasmid; wherein said plasmid comprises: a nucleotide sequence comprising at least one sequence selected from a group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, and SEQ ID NO: 07; and a regulatory element.
Preferably, said regulatory element comprises a promoter and a ribosome binding site.
Preferably, said plasmid is pET-MSY, pET-YjgD, pET-D, or pET-SUMO.
Preferably, said plasmid further comprises a gene encoding a fusion partner.
Preferably, said fusion partner is msyB of E. coli, yjgD of E. coli, protein D of Lambda bacteriophage, or SUMO of S. cerevisiae. 
Preferably, said expression vector is used for an E. coli gene expression system.
To sum up, the present invention is related to antigens that are suitable for being used as the active ingredient of a M. hyopneumoniae subunit vaccine and a M. hyopneumoniae subunit vaccine/composition prepared by using the same. The present subunit vaccine not only can be effectively used in prevention task for lowering down the cost thereof, the disclosure of the present invention also shows that a “cocktail” subunit vaccine (i.e. having at least two antigens as active ingredients) using at least two antigens of the present invention has improved induction of immune response.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.
FIG. 1 shows the result of the two-dimensional gel protein electrophoresis conducted in the 1st example of the present invention.
FIG. 2 shows the result of the color reaction of the Western blot conducted in the 1st example of the present invention.
FIG. 3 shows the result of the electrophoresis of the PCR products obtained in the 2nd example of the present invention.
FIG. 4 shows the records of the challenge experiments conducted in the 3rd example of the present invention.



DESCRIPTION OF REFERENCE SIGNS IN THE FIGURES


    
    
        1 XylF(xylose-binding lipoprotein)
        2 XylF(xylose-binding lipoprotein)
        3 XylF(xylose-binding lipoprotein)
        4 PdhA(pyruvate dehydrogenase E1-alpha subunit)
        5 Mhp145(periplasmic sugar-binding protein)
        6 EutD(phosphotransacetylase)
        7 EutD(phosphotransacetylase)
        8 Mhp389
        9 P78(lipoprotein)
        10 P132
    
    


DETAILED DESCRIPTION OF THE INVENTION
One of the core concepts of the present invention is to survey potential candidate antigens suitable for subunit vaccines by using two-dimensional gel protein electrophoresis along with immunological screening technology and to identify the antigens by mass spectrometer. Then, the performance of the present subunit vaccines were verified by animal model experiments.
Briefly, the progress of the development of the present invention is:
(1) Inducing immune response of experiment pigs by injecting a conventional M. hyopneumoniae vaccine and obtaining serum containing anti-M. hyopneumoniae antibodies. (2) Obtaining total proteins of M. hyopneumoniae for two-dimensional gel protein electrophoresis. (3) Conducting hybridization of the result of the two-dimensional gel protein electrophoresis of step (2) by using the serum of step (1) as 1st antibody, and then collecting proteins showing positive (i.e. candidate antigens) from the gel after amplification by a 2nd antibody and the following development procedure. (4) Identifying the candidate antigens obtained in step (3). (5) Expressing said candidate antigens in large amounts by using an E. coli gene expression system. (6) Examining the efficacy of the present subunit vaccines in reducing pathological traits in lung by swine challenge experiments and thereby verifying the value of said candidate antigens in being used as active ingredient of a subunit vaccine.
The present vaccine for preventing Mycoplasma spp. infection comprises an active ingredient and a pharmaceutically acceptable adjuvant.
In an embodiment of the present invention, said active ingredient may be PdhA, XylF, EutD, Mhp145, P78, P132, or Mhp389. In an alternative embodiment, as long as the antigenic determinant of any of the aforesaid protein is not interfered, said active ingredient may be a fusion protein of any two of the aforesaid proteins. In another alternative embodiment, said active ingredient comprises at least two of the aforesaid proteins; that is, so called a “cocktail” vaccine of the present invention.
In another embodiment of the present invention, said active ingredient may comprise an amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a combination thereof. In an alternative embodiment, as long as the antigenic determinant formed by folding of a peptide of said amino acid sequence is not interfered, said active ingredient may be a fusion protein with at least two said sequences. In another alternative embodiment, said active ingredient comprises two or more proteins respectively comprising one of the aforesaid amino acid sequences; that is, so called a “cocktail” vaccine of the present invention.
Said pharmaceutically acceptable adjuvant is used for improving the immune effect of said active ingredient, stabilizing said active ingredient, and/or increasing the safety of vaccines. Said pharmaceutically acceptable adjuvant of the present invention includes, but not limits to: a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.
The vaccine of the present invention may have one or at least two said active ingredients (i.e. a cocktail vaccine). In an example of the present vaccine, said active ingredient is of a concentration of 50 to 3500 μg/mL based on the total volume of said vaccine. In a preferable embodiment of the present invention, when said vaccine comprises only one said active ingredient, said active ingredient is of a concentration of 50 to 500 μg/mL based on the total volume of said vaccine. In an alternative embodiment of the present invention, the present vaccine comprises at least one said active ingredient; wherein the total concentration of said active ingredient(s) contained in said vaccine is 50 to 1000 μg/mL, 50 to 1500 μg/mL, 50 to 2000 μg/mL, 50 to 2500 μg/mL, 50 to 3000 μg/mL, or 50 to 3500 μg/mL based on the total volume of said vaccine.
Another aspect of the present invention is to provide an expression vector for preventing Mycoplasma spp. infection. Specifically, said expression vector may be used for an E. coli gene expression system. Nevertheless, without being apart from the spirit of the present invention, those having ordinary skill in the art can modify said vector based on the disclosure of the present invention and make said vector suitable for different gene expression system while still belongs to the scope of the present invention.
Said expression vector comprises a plasmid. Said plasmid comprises: a nucleotide sequence comprising at least one sequence selected from a group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, and a combination thereof; and a regulatory element.
Said vector is used in an E. coli gene expression system and for producing the antigens of the present invention via E. coli. In other words, said nucleotide sequence can be translated into the amino sequence of the present antigen via an E. coli gene expression system and then the amino acid sequence can fold into the present antigen.
In an alternative embodiment, as long as the operation of the E. coli gene expression system is not hindered and the production of said nucleotide sequence and the folding of the consequent amino acid sequence thereof are not interfered, said plasmid may comprise two or more said nucleotide sequences.
Said regulatory element is referred to an element required for initiating the transcription and translation in the expression system. Said regulatory element shall at least comprise a promoter, and a ribosome binding site. Preferably, said regulatory element may further comprise: an operator, an enhancer sequence, or a combination thereof.
In a preferable embodiment of the present invention, said plasmid further comprises a gene encoding a fusion partner. Said fusion partner includes but not limits to msyB of E. coli, yjgD of E. coli, protein D of Lambda bacteriophage, or SUMO of S. cerevisiae. Said MsyB is rich in acidic amino acid and might be favorable for improving the solubility of the proteins to be produced.
The following examples recite the trials and experiments of the present invention in order to further explain the features and advantages of the present invention. It shall be noted that the following examples are exemplary and shall not be used for limiting the claim scope of the present invention.
Example 1: Screening for Candidate Antigens Suitable for being Used as Active Ingredient of a Subunit Vaccine
Preparation of Serum Containing Anti-Swine Mycoplasm spp. Antibody
According to researches, there are seven Mycoplasm spp. can be isolated from swine: Mycoplasm hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma flocculare, Mycoplasma hyopharyngis, Mycoplasma sualvi, Mycoplasma bovigenitalium (Gourlay et al., 1978; Blank et al., 1996; Assuncao et al., 2005). Among them, M. hyopneumoniae is the major pathogen of swine enzootic pneumonia with an infection rate of 25 to 93%. Therefore, the present invention used M. hyopneumoniae (PRIT-5 strain) for immune proteomics studies and as sources of genes encoding antigens. Friis medium (Friis et al., 1975) as used for culturing M. hyopneumoniae. According to the experiment design, a proper amount of antibiotic or agar of 1.5% was added to formulating a solid medium.
Three SPF pigs of 4-week old were brought from Agricultural Technology Research Institute and fed with same feed and kept at same environment and growth condition in piggery before experiments.
After the pigs were fed to 32-day, 46-day, and 60-day old, the pigs were administrated 2 mL of Bayovac® MH-PRIT-5 (M. hyopneumoniae PRIT-5) vaccine via intramuscular injection. Then, the pigs were continuously fed to 74-day old and blood was collected from a jugular vein thereof. The collected blood was placed in room temperature for 1 hour and stored in 4° C. In the next day, the collected blood was centrifugated at 1,107×g for 30 minutes and the supernatant was removed to a clean tube and stored in −20° C.
Two-Dimensional Gel Protein Electrophoresis of the Total Protein of Mycoplasm spp.
ReadyPrepTM protein extraction kit (total protein) (Bio-Rad, CA, USA) was used for extracting the total protein of Mycoplasm spp. Afterward, the concentration of the protein collected was determined by using a Bio-Rad RC DC Protein Assay Kit (CA, USA). The detailed protocol can be referred from the product description or can be modified from well-known protocols in the field.
The two-dimensional gel protein electrophoresis was conducted in two steps: isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IEF was to separate proteins in the sample in view of isoelectric point thereof. SDS-PAGE was to separate proteins accordance with molecular weight thereof. Please see FIG. 1, which shows the result of the two-dimensional gel protein electrophoresis.
Hybridization
The serum obtained in step (1) was used as 1st antibody to hybridize with the result of the two-dimensional gel protein electrophoresis in step (2). After being amplified by 2nd antibody and developed by the following development procedure, proteins showing positive were collected. Those proteins were recognized by the anti-Mycoplasm spp. antibody and therefore would be suitable as candidate antigens for active ingredient of subunit vaccines.
The hybridization was conducted by Western blotting. Briefly, the 2D gel after electrophoresis was transferred to a PVDF membrane. Then, the membrane was incubated and hybridized sequentially with 1st antibody (the serum containing anti-Mycoplasm spp. antibody) and 2nd antibody (AP-conjugated anti-pig IgG). Afterward, a color reaction was conducted by using NBT/BCIP solution.
The result of the color reaction of Western blotting was shown in FIG. 2; wherein 10 proteins positive to the immuno-hybridization with anti-Mycoplasm spp. antibody were marked as candidate antigens for being used as active ingredients of subunit vaccines.
Identification of the Candidate Antigens Obtained
According to the color reaction of the Western blotting, the gel corresponding to the positive location on the membrane was cut by micropeptide and analyzed by mass spectrometry. The obtained data of the mass spectrometry was then matched with amino acid sequence and protein database to identify those proteins.
Please see the following table 1, said 10 proteins positive to the immune-hybridization with anti-Mycoplasm spp. antibody were listed.







TABLE 1







the 10 proteins positive to the immune-hybridization with


anti-Mycoplasm spp. antibody and amino sequence thereof.









Candidate
Name
SEQ ID NO












1
XylF (xylose-binding lipoprotein)
SEQ ID NO: 09


2
XylF (xylose-binding lipoprotein)
SEQ ID NO: 09


3
XylF (xylose-binding lipoprotein)
SEQ ID NO: 09


4
PdhA (pyruvate dehydrogenase E1-alpha
SEQ ID NO: 08



subunit)



5
Mhp145 (periplasmic sugar-binding
SEQ ID NO: 11



protein)



6
EutD (phosphotransacetylase)
SEQ ID NO: 10


7
EutD (phosphotransacelylase)
SEQ ID NO: 10


8
Mhp389
SEQ ID NO: 14


9
P78 (lipoprotein)
SEQ ID NO: 12


10
P132
SEQ ID NO: 13





*XylF and EutD have different charge states in cells and therefore become 3 and 2 positive location on the membrane.






Example 2: Expressing of Said Candidate Antigens in Large Amount by E. coli Gene Expression System
Escherichia coli JM109 was used as the host cells for cloning and Escherichia coli BL21 (DE3) was used as the host cells for protein expression. The Escherichia coli cells were cultured in LB medium (Luria-Bertani; Difco, Mich., USA). According to the experiment design, a proper amount of antibiotic or agar of 1.5% was added to formulating a solid medium.
Amplification of the Genes Encoding the Candidate Antigens
After the candidate antigens were identified, the genes encoding those antigens were searched in the NCBI database (National Center for Biotechnology Information). Specific primers targeting the antigen genes were designed accordingly. Then, the antigen genes were amplified by using the specific primers and the chromosome of M. hyopneumoniae PRIT-5 as template. The specific primers used were listed in the following table 2.







TABLE 2







Primer set.










Candidate
Sequences of the primer set






PdhA
PdhAF (SEQ ID NO: 15)




5′-GATATAGGATCCATGGACAAATTTCGCTATG 




TAAAGCCT G-3′ PdhAR (SEQ ID NO: 16)




5′-CAATATGTCGACTTATTTTACTCCTTTAAAA




AATTCAAGCGCTTC-3′



XylF
XylFF (SEQ ID NO: 17)




5′-GATATAGGATCCATGAATGGAATAAATTTCT




TGGCTTAGGCTTAGTTTTTC-3′




Xy1FR (SEQ ID NO: 18)




5′-CAATATGTCGACTTAATTTTTATTAATATCG




GTAATTAGTTTGTCTAAGC-3′



EutD
EUTDF (SEQ ID NO: 19)




5′-GATATAGGATCCATGACATACCAAGAATATC




TTCAAGCAA G-3′)




EUTDR (SEQ ID NO: 20)




5′-CAATATGTCGACCTATTTACCTTCTTCAAC




TTGTAGAGCGCT-3′)



Mhp145
Mhp145F (SEQ ID NO: 21)




5′-GATATAGGATCCATAGCTTCAAGGTCGAA 




TACAACTGC-3′ Mhp145R (SEQ ID NO: 22)




5′-CAATATGTCGACTTAATTTACCTTTTGGAG 




TATCCCATTTTC-3′



P78
P78F (SEQ ID NO: 23)




5′-GATATAGGATCCTTATCCTATAAATTTAGG 




CGTTTTTTCC-3′ P78R (SEQ ID NO: 24)




5′-CAATATGTCGACTTATTTTGATTTAAAAGCA 




GGACCTAA AT-3′



P132
PI32F (SEQ ID NO: 25)




5′-GATATAGGATCCATTGGACTAACAATTTTTG




AGAAATCATTTAG-3′




P132R (SEQ ID NO: 26)




5′-CAATATGTCGACTTATTCCTAAATAGCCCC 




ATAAAGTG-3′



Mhp389
Mhp389F (SEQ ID NO: 27)




5′-GATATAGGATCCATGGACAAATTTTCACGA 




ACTGTTCT-3′ Mhp389R (SEQ ID NO: 28)




5′-CAATATGTCGACCTAGATTTTAAAGGATTTTT




TTAATTCAATAATATAATC-3′









Polymerase chain reaction (PCR) was conducted with the primer sets listed in the table 2 above to amplify the genes of the candidate antigens. The amplified genes were then used in the E. coli gene expression system. The PCR condition was: 5 minutes in 98° C. (one round); 30 seconds in 94° C., 30 seconds in 55° C., X seconds in 68° C. (35 rounds); 5 minutes in 68° C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. After the PCR reaction, an electrophoresis was conducted to verify if the PCR products contained the DNA fragments of expected size. Please see FIG. 3, which shows the electrophoresis result of the PCR products; wherein lane 1 was eutD gene; lane 2 was pdhA; lane 3 was xylF; lane 4 was P78 gene; lane 5 was P132 gene; lane 6 was mhp145; lane 7 was mhp389.
Cloning of the PCR Products
The cloning was conducted by using a CloneJET PCR Cloning Kit, and the ligation mixture was transformed into E. coli ECOSTM 9-5 (Yeastern, Taipei, Taiwan). The detailed protocol can be referred from the product description or modified from the well-known protocol in the field.
After transformation, the bacteria were cultured on a solid LB medium containing ampicillin (100 μg/mL) until colony thereof formed. Then, colony PCR was conducted to screen strains success in transformation. The PCR condition was: 5 minutes in 95° C. (one round); 30 seconds in 95° C., 30 seconds in 55° C., X seconds in 72° C. (25 rounds); 7 minutes in 72° C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of Taq DNA polymerase (Genomics, Taipei, Taiwan) is 1 kb/min; therefore, if Taq DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 1 minute.
The plasmids of strains, whose recombinant plasmids were verified having the insert DNA, were then proceeded to DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids containing eutD, pdhA, xylF, P78 gene, P132 gene, mhp145, and mhp389 were named as pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132, pJET-mhp145, pJET-mhp389, respectively.
Point Mutation and Cloning of the Antigen Genes of M. hyopneumoniae 
Before amplifying the candidate antigens in an E. coli gene expression system, the codon usage in different organisms shall be considered. That said, if the gene contains codon that would be encoded ambiguously between the original organism therefrom and E. coli, the gene shall be modified by point mutation.
The M. hyopneumoniae antigen genes, pdhA, xylF, P78 gene, P132 gene, mhp145, and mhp389, contain TGA codon (eutD does not have the concern in codon usage like others). The TGA codon was translated into tryptophan in Mycoplasma spp. but translated as stop codon in E. coli. In order to prevent from not being able to produce the entire protein in an E. coli gene expression system, primers targeting the TGA site were designed and point mutation replacing TGA with TGG was conducted by using overlapping extension polymerase chain reaction. As a result, the genes to be expressed in the E. coli gene expression system can be truthfully translated into the candidate antigen of the present invention. Besides, the cutting sites of BamHI of P78 gene, P132 gene, and mhp389 were undergone silent mutation for the convenience of cloning.
The primers used for point mutation was designed to locate the site of point mutation at the central part of the primer and to have a Tm value of higher than 78° C. The Tm value of the primers for point mutation was calculated by using the formula provided by Invitrogene Co.: Tm=81.5+0.41 (% GC)−675/N−% mismatch; wherein % GC is referred as the percentage of GC in view of the total nucleotides contained in the primer concerned; N is referred as the length of the primer concerned; % mismatch is referred as the percentage of the base to be mutated in view of the total nucleotides contained in the primer concerned. The primer sets used for the aforesaid genes were listed in the following Table 3 to Table 8.







TABLE 3







The primer sets for point mutation of pdhA.








Primer
DNA sequence (5′ to 3′)





Pd hAF
GATATAGGATCCATGGACAAATTTCGCTATGTAAAG


SEQ ID NO: 29
CCTG


PdhAM1
GCTAACAAAAGATGACTGGTTTGTCCCAGCTTTTCG


SEQ ID NO: 30



PdhAM2
CGAAAAGCTGGGACAAACCAGTCATCTTTTGTTAGC


SEQ ID NO: 31



PdhAM3
CTTGCAAATGCAATATTGGAATGGTAGCGAAAAAGG


SEQ ID NO: 32



PdhAM4
CCTTTTTCGCTACCATTCCAATATTGCATTTGCAAG


SEQ ID NO: 33



PdhAM5
CGAGGCGCTAAATATTGCAAGTATTTGGAAATGGCC


SEQ ID NO: 34 
AGTTGTTTTTTGCGTAAATAAC


PdhAM6
GTTATTTACGCAAAAAACAACTGGCCATTTCCAAAT


SEQ ID NO: 35 
ACTTGCAATATTTAGCGCCTCG


PdhAM7
GTTTTTTGCGTAAATAACAATCAATGGGCAATTTCA


SEQ ID NO: 36 
ACCCCAAATAAATATG


PdhAM8
CATATTTATTTGGGGTTGAAATTGCCCATTGATTGT


SEQ ID NO: 37 
TATTTACGCAAAAAAC


PdhAM9
GTTGAGTTTGTAACTTGGCGTCAAGGTGTTCATACC


SEQ ID NO: 38



PdhAM10
GGTATGAACACCTTGACGCCAAGTTACAAACTCAAC


SEQ ID NO: 39



PdhAM11
GAGAACACGAAAAATGGGAACCAATGCACCGG


SEQ ID NO: 40



PdhAM12
CCGGTGCATTGGTTCCCATTTTTCGTGTTCTC


SEQ ID NO: 41



PdhAM13
CCGAAAAACAAAAAATTTGGGATGAAGCGCTTGCGA


SEQ ID NO: 42
TTG


PdhAM14
CAATCGCAAGCGCTTCATCCCAAATTTTTTGTTTTT


SEQ ID NO: 43
CGG


PdhAR
CAATATGTCGACTTATTTTACTCCTTTAAAAAATTC


SEQ ID NO: 44
AAGCGCTTC
















TABLE 4







The primer sets for point mutation of xylF.








Primer
DNA sequence (5′ to 3′)





XylFF
GATATAGGATCCATGAAATGGAATAAATTTCTTGGC


SEQ ID NO: 45 
TTAGGCTTAGTTTTTC


Xy1FM1
CATTTAACCAATCAAGTTGGGAGGCAATTCAACAAC


SEQ ID NO: 46
TTGG


Xy1FM2
CCAAGTTGTTGAATTGCCTCCCAACTTGATTGGTTA


SEQ ID NO: 47
AATG


XyIFM3
CTAATACCAACAAAAATGTTTGGGTACTTTCTGGTT


SEQ ID NO: 48 
TTCAACACG


Xy1FM4
CGTGTTGAAAACCAGAAAGTACCCAAACATTTTTGT


SEQ ID NO: 49 
TGGTATTAG


XylFM5
CGGTGATGCGATCACAAAATGGTTAAAAATCCCTGA


SEQ ID NO: 50 
AAATAAGC


XylFM6
GCTTATTTTCAGGGATTTTTAACCATTTTGTGATCG


SEQ ID NO: 51
CATCACCG


Xy1FM7
TTATCATACTCGGAATTGACTGGACTGATACTGAAA


SEQ ID NO: 52 
ATGTAATTC


XylFM8
GAATTACATTTTCAGTATCAGTCCAGTCAATTCCGA


SEQ ID NO: 53 
GTATGATAA


Xy1FM9
GAAGAAGCCGGATGGCTTGCAGGATATGC


SEQ ID NO: 54



Xy1FM10
GCATATCCTGCAAGCCATCCGGCTTCTTC


SEQ ID NO: 55



XylFM11
GGTTATCTAGCCGGAATTAAAGCTTGGAATCTAAAA


SEQ ID NO: 56 
AATTCTGATAAAAAAAC


Xy1FM12
GTTTTTTTATCAGAATTTTTTAGATTCCAAGCTTTA


SEQ ID NO: 57 
ATTCCGGCTAGATAACC


XylFR
CAATATGTCGACTTAATTTTTATTAATATCGGTAAT


SEQ ID NO: 58 
TAGTTTGTCTAAGC
















TABLE 5







The primer sets for point mutation of P78 gene.








Primer
DNA sequence (5′ to 3′)





P78F
GATATAGGATCCTTATCCTATAAATTTAGGCGTTTT


SEQ ID NO: 59
TTCC


P78M1
CAATTAATAAAGTTTTGTTTGGTTGGATGATTAATA


SEQ ID NO: 60 
AAGCACTTGCTGATCC


P78M2
GGATCAGCAAGTGCTTTATTAATCATCCAACCAAAC


SEQ ID NO: 61 
AAAACTTTATTAATTG


P78M3
GATATTAAAGAAATTGAAAGAATCTGGAAAAAATAT


SEQ ID NO: 62 
GTCTCCGATGATCAAGG


P78M4
CCTTGATCATCGGAGACATATTTTTTCCAGATTCTT


SEQ ID NO: 63 
TCAATTTCTTTAATATC


P78M5
GCCCTTTCAGGAGGCTCCACTGATTCGGCA


SEQ ID NO: 64



P78M6
TGCCGAATCAGTGGAGCCTCCTGAAAGGGC


SEQ ID NO: 65



P78M7
GCCGCAAAAGCTTTTGTTAAATGGCTTTTGACAGAA


SEQ ID NO: 66 
AAAATAGTCT


P78M8
AGACTATTTTTTCTGTCAAAAGCCATTTAACAAAAG


SEQ ID NO: 67 
CTTTTGCGGC


P78R
CAATATGTCGACTTATTTTGATTTAAAAGCAGGACC


SEQ ID NO: 68
TAAAT
















TABLE 6







The primer sets for point mutation of P132 gene.








Primer
DNA sequence (5′ to 3′)





P132F
GATATAGGATCCATTGGACTAACAATTTTTGAGAAA


SEQ ID NO: 69 
TCATTTAG


P132M1
CTAACTTCTCTAAAAGGTTGGAAAGAAGAAGATGAT


SEQ ID NO: 70
TTTG


P132M2
CAAAATCATCTTCTTCTTTCCAACCTTTTAGAGAAG


SEQ ID NO: 71
TTAG


P132M3
CTTTCTATTACTTTTGAACTCTGGGACCCAAATGGT


SEQ ID NO: 72 
AAATTAGTATC


P132M4
GATACTAATTTACCATTTGGGTCCCAGAGTTCAAAA


SEQ ID NO: 73 
GTAATAGAAAG


P132M5
CCCTGAAGGAGATTGGATAACTTTAGGGAG


SEQ ID NO: 74



P132M6
CTCCCTAAAGTTATCCAATCTCCTTCAGGG


SEQ ID NO: 75



P132M7
CTACCAGGAACTACCTGGGATTTCCATGTTGAAC


SEQ ID NO: 76



P132M8
GTTCAACATGGAAATCCCAGGTAGTTCCTGGTAG


SEQ ID NO: 77



P132M9
GGACAACTAATTTGGAGCCAGTTAGCTTCC


SEQ ID NO: 78



P132M10
GGAAGCTAACTGGCTCCAAATTAGTTGTCC


SEQ ID NO: 79



P132M11
GGAACAAAAAAGGAATGGATTCTTGTAGGATCTGG


SEQ ID NO: 80



P132M12
CCAGATCCTACAAGAATCCATTCCTTTTTTGTTCC


SEQ ID NO: 81



P132M13
CCAATACGCAAATATGGATAACCCGTCTAGGAAC


SEQ ID NO: 82



P132M14
GTTCCTAGACGGGTTATCCATATTTGCGTATTGG


SEQ ID NO: 83



P132M15
CCAAGGGGAAGTTCTCTGGACTACTATTAAATCCAA


SEQ ID NO: 84
AC


P132M16
GTTTGGATTTAATAGTAGTCCAGAGAACTTCCCCTT


SEQ ID NO: 85
GG


PI32M17
CAAAAAACTTCACCTTTGGTGGATTGCTAATGATAG


SEQ ID NO: 86
C


P132M18
GCTATCATTAGCAATCCACCAAAGGTGAAGTTTTTT


SEQ ID NO: 87
G









P132R
CAATATGTCGACT
TATTCCTAAATAGCCCCATAAAG








SEQ ID NO: 88
TG
















TABLE 7







The primer sets for point mutation of mhp145.








Primer
DNA sequence (5′ to 3′)





Mhp145F
GATATAGG ATCCAT AGCTTCAAGGTCGAATACAA


SEQ ID NO: 89
CTGC


Mhp145M1
AATAATTGCAGAAAAAATTCTTAAAGATCAATGGAA


SEQ ID NO: 90
AACAAGTAAATATTCTGATTTTTATTCACAAT


Mhp145M2
ATTGTGAATAAAAATCAGAATATTTACTTGTTTTCC


SEQ ID NO: 91
ATTGATCTTTAAGAATTTTTTCTGCAATTATT


Mhp145R
CAATATGTCGACTTA ATTTACCTTTTGGAGTATCC


SEQ ID NO: 92
CATTTTC
















TABLE 8







The primer sets for point mutation of mhp389.








Primer
DNA sequence (5′ to 3′)





Mhp389F
GATATAGGATCCATGGACAAATTTTCACGAACTG


SEQ ID NO: 93
TTCT


Mhp389M1
CAATAGTGACAATGGACCCCCCAAATGTTGGTCG


SEQ ID NO: 94



Mhp389M2
CGACCAACATTTGGGGGGTCCATTGTCACTATTG


SEQ ID NO: 95



Mhp389M3
GATAAAGGCGCATCATGGCTTGCGCTTGCACCAAC


SEQ ID NO: 96



Mhp389M4
GTTGGTGCAAGCGCAAGCCATGATGCGCCTTTATC


SEQ ID NO: 97



Mhp389M5
GGAAAACTTAAAGGTAAATGGACTTTTGGACTAA


SEQ ID NO: 98
CCTATTT


Mhp389M6
AAATAGGTTAGTCCAAAAGTCCATTTACCTTTAA


SEQ ID NO: 99
GTTTTCC


Mhp389R
CAATATGTCGACCTAGATTTTAAAGGATTTTTTT


SEQ ID NO: 100
AATTCAATAATATAATC









The method for the point mutation was briefly explained as follows. The chromosome of M. hyopneumoniae PRIT-5 was used as template and DNA fragments was amplified by using the primer sets set forth in the table 3 to table 8 above.
The 50 μL PCR reaction mixture comprised 1×GDP-HiFi PCR buffer, 200 μM of mixture of dATP, dTTP, dGTP, and dCTP, 1 μM of primers, 100 ng of chromosome of M. hyopneumoniae PRIT-5, and 1 U of GDP-HiFi DNA polymerase. The PCR condition was: 5 minutes in 98° C. (one round); 30 seconds in 94° C., 30 seconds in 55° C., X seconds in 68° C. (35 rounds); 5 minutes in 68° C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of GDP-HIFI DNA polymerase (GeneDirex, Las Vegas, USA) is 1 kb/15 seconds; therefore, if GDP-HIFI DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 15 seconds. After the PCR reaction, an electrophoresis was conducted to verify if the PCR products contained the DNA fragments of expected size. Then, the PCR product was recycled by using a Gel-MTM gel extraction system kit.
Afterward, the PCR product was used as template and amplified by using the primer sets set forth in the table 2 above. The PCR condition was: 2 minutes in 98° C. (one round); 30 seconds in 94° C., 30 seconds in 55° C., X seconds in 68° C. (35 rounds); 5 minutes in 68° C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of GDP-HIFI DNA polymerase (GeneDirex, Las Vegas, USA) is 1 kb/15 seconds; therefore, if GDP-HIFI DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 15 seconds. After the aforesaid amplification step, a full length sequence of the candidate antigen genes with point mutation can be obtained.
Then, the PCR product was recycled by using a PCR-MTM Clean Up system kit (GeneMark, Taichung, Taiwan) and the cloning thereof was conducted by using a CloneJET PCR Cloning Kit. Colony PCR was conducted to confirm the strains after transformation containing plasmid having the insert DNA and then the plasmids therein were isolated for DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids containing mutated candidate antigen genes were named as pJET-pdhAM, pJET-xylFM, pJET-P78M, pJET-P132M, pJET-mhp145M, pJET-mhp389M, respectively.
According to the result of sequencing, the DNA sequences of the candidate antigen genes after point mutation were as shown in SEQ ID NO:01 (pdhA), SEQ ID NO:02 (xylF), SEQ ID NO:03 (eutD, was not point-mutated), SEQ ID NO:04 (mhp145), SEQ ID NO:05 (P78 gene), SEQ ID NO:06 (P132 gene), SEQ ID NO:07 (mhp389).
Construction of the Expression Vectors for Expressing the M. hyopneumoniae Antigens
In this part of experiments, plasmid pET-MSY was used as backbone for constructing an expression vector for expressing M. hyopneumoniae antigen. pET-MSY is a derivative of pET29a and has a E. coli msyB. Therefore, the expressed recombinant antigen thereby would have a fusion partner MsyB. MsyB is rich in acidic amino acid and is able of increasing the solubility of the protein expressed.
After pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132, pJET-mhp145 and pJET-mhp389 being digested by BamHI and SalI, DNA fragment obtained was inserted into pET-Msy digested previously with the same restriction enzymes by ligase. Then, the pET-Msy with the DNA fragment was transformed into E. coli ECOS 9-5. Colony PCR was conducted to confirm the strains after transformation containing plasmid having the insert DNA and then the plasmids therein were isolated for DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids verified with correct DNA sequence were named as pET-MSYEutD, pET-MSYPdhA, pET-MSYXylF, pET-MSYP78, pET-MSYP132, pET-MSYMhp145, and pET-MSYMhp389, respectively. Those plasmids obtained were examples of the expression vectors for preventing Mycoplasma spp. infection of the present invention.
Expression and Isolation of the M. hyopneumoniae Antigens
The vectors for antigen expression were transformed into E. coli BL21 (DE3). Single colony of consequent strains after transformation was inoculated in LB liquid medium containing kanamycin (working concentration: 30 μg/mL). After culture overnight at 37° C., 180 rpm, the suspension of the bacteria was diluted at ratio of 1:100 and inoculated again in another LB liquid medium containing kanamycin (working concentration: 30 μg/mL). The bacteria were cultured at 37° C., 180 rpm until OD600 therefore achieving about 0.6 to 0.8. Then, 0.1 mM of IPTG was added to induce expression. After induction for 4 hours, pellet was collected by centrifugation (10000×g, 10 minutes, 4° C.) and the expression was examined via protein electrophoresis.
Afterward, immobilized-metal affinity chromatography (IMAC) was used for protein isolation through the covalent bonding between the His tag of the N-terminal of the recombinant protein and nickel ions or cobalt ions. The protocol of protein isolation was in accordance with the product description of the QIAexpressionistTM (fourth edition, Qiagen). The pellet was suspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and disturbed by an ultrasonic processor. After centrifugation (8,000×g, 15 minutes), the supernatant was collected to introduce into a column of 1 mL Ni-NTA resin. The recombinant antigens would adhere on said resin. Then, 15 mL wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) was introduced into the column to wash the resin so that nonspecific proteins adhering thereon can be removed. Lastly, 20 mL elution buffer was added (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) to wash off the recombinant antigens on the resin; wherein the imidazole of high concentration can compete the binding site on the resin with the recombinant proteins and thereby cause the recombinant proteins being washed off. The result of isolation was then examined by protein electrophoresis.
The candidate antigens of the present invention collected by isolation can then be used for the following immune trials to confirm their ability to be used as active ingredient of anti-Mycoplasm spp. subunit vaccines.
Example 3: Swine Immune Challenge Experiments of the Candidate Antigens of the Present Invention
In this example, the candidate antigens of the present invention were used as active ingredient for preparing subunit vaccines and tested for immune effects thereof in live swine.
Vaccine Preparation
One isolated recombinant antigen or several isolated recombinant antigens were mixed with alumina gel as an adjuvant to prepare a subunit vaccine or a cocktail subunit vaccine. Every dose of the prepared vaccine was of 2 mL in volume and each kind of antigen contained therein was of 100 μg.
The following table 9 listed the samples prepared in this example for immune challenge experiments.







TABLE 9







Samples of vaccine prepared in Example 3








Sample
Active Ingredient (Antigen)





1
PdhA


2
XylF


3
EutD


4
Mhp145


5
P78


6
P132


7
Mhp389


8
PdhA + P78


9
XylF + Mhp145









The swine immune challenge experiments would be conducted by using Bayovac® MH-PRIT-5 (made by using M. hyopneumoniae PRIT-5, as a positive control group), subunit vaccines (samples 1-7 of the present invention), and cocktail vaccines (samples 8 and 9 of the present invention).
33 SPF pigs of 4-week old were brought from Agricultural Technology Research Institute and fed with same feed, environment, and growth condition in piggery before experiments.
After the pigs were fed to 35-day and 49-day old, the pigs were administrated 2 mL of vaccine above via intramuscular injection.
Challenge Experiments
The aforesaid pigs being induced immune response were challenged by Mycoplasm spp. at 109-day old to confirm the immune effect of the aforesaid vaccines.
First of all, a lung collected from pigs infected by Mycoplasm spp. was ground in 20 mL of Friis medium and centrifugated at 148.8×g for 10 minutes. The supernatant was removed to a clean tube and centrifugated again at 7,870×g for 40 minutes. Then, the supernatant was discarded and the precipitation was suspended in 6 mL of Friis medium to obtain a suspension. Afterward, the suspension was filtered by membrane of 5 μm and 0.45 μm sequentially to obtain bacteria solutions required for the challenge experiments.
The bacteria solution (5 mL) was administrated to narcotized pigs via trachea thereof. After 28 days from administration, the pigs were sacrificed and dissected to collect lung thereof. The immune effect was examined by observing the lung and recorded according to the following criteria: any of meddle upper lobes and upper lobes of any side of the lung observed of pathological trait was scored as 10 points; any of meddle upper lobe and diaphragmatic lobes of any side of the lung observed of pathological trait was scored as 5 points. The full score was 55 points. The observation records were shown in FIG. 4.
In comparison with the results of non-injected pigs, the seven candidate antigens of the present invention were able to provide equivalent immune effects as conventional vaccine (Bayovac® MH-PRIT-5). If the higher safety of subunit vaccines is taking into consideration, the vaccines containing the candidate antigens of the present invention shall be valued more.
On the other hand, it was not common to use two or more antigens that would induce immune effects in one vaccine because the two or more antigens may not provide doubled immune effect. In fact, there is higher chance that the two or more antigens may interfere or against each other and consequently reduce the immune effect of the vaccine. According to the result of this example, sample 8 and sample 9 of the present invention (i.e. cocktail vaccine) unexpectedly provide significant increase in the immune effect. That said, the subunit vaccines of the present invention not only have high safety but also provide better immune effect when the candidate antigens of the present invention are used in combination.
Those having ordinary skill in the art can readily understand any possible modifications based on the disclosure of the present invention without apart from the spirit of the present invention. Therefore, the examples above shall not be used for limiting the present invention but intend to cover any possible modifications under the spirit and scope of the present invention according to the claims recited hereinafter.

## Claims

1. A composition for preventing a disease caused by Mycoplasma spp., comprising:
an active ingredient, comprising a protein of Mhp389; and
a pharmaceutically acceptable adjuvant;
wherein said Mhp389 comprises the sequence of SEQ ID NO: 14.

2. The composition of claim 1, wherein said active ingredient is of a concentration of 50 to 3500 μg/mL based on the total volume of said composition.

3. The composition of claim 1, wherein said pharmaceutically acceptable adjuvant is a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.

4. The composition of claim 1, further comprising a pharmaceutically acceptable additive.

5. The composition of claim 4, wherein said pharmaceutically acceptable additive is a solvent, a stabilizer, a diluent, a preservative, an antibacterial agent, an antifungal agent, an isotonic agent, a absorption delaying agent, or a combination thereof.