JP2010193759A - Oral administration-type multivalent vaccine for swine erysipelas and swine mycoplasmal pneumonia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and economical vaccine effective for phylaxis of swine erysipelas and mycoplasma hyopneumoniae even by oral administration. <P>SOLUTION: The Koganei strain of Erysipelothrix rhusiopathiae expressing a part of mycoplasma hyopneumoniae P97 adhesin is created by selecting the Koganei strain of the Erysipelothrix rhusiopathiae long time-proven as a live vaccine for injection in our country as an attenuated Erysipelothrix rhusiopathiae as a vector for the oral administration-type mycoplasma vaccine, and introducing a part of the P97 adhesin gene of the mycoplasma hyopneumoniae to the bacterium by homologous recombination. The strain exhibits good vaccine effects on the strong poison swine erysipelas bacteria and the strong poison mycoplasma hyopneumoniae. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an invention of swine erysipelas / pig mycoplasma pneumonia oral administration type mycoplasma vaccine. The present invention relates to a mycoplasma hyopneumoniae adhesin protein (P97) and a polypeptide comprising a repetitive sequence in the paralog, a gene encoding the polypeptide, and use thereof as a vaccine for protection against swine mycoplasma pneumonia infection About.

  In the recent large-scale pig farming industry, control of respiratory diseases of pigs is one of the indispensable elements for management. Swine respiratory disease has become increasingly complex as the study progresses. In other words, it became clear that several viruses and bacteria work in combination to cause respiratory diseases, and the concept of “composite respiratory diseases in pigs” has become common. Although disease control has been carried out mainly with vaccines and antibiotics, in recent years there has been a tendency to limit the use of antibiotics, and the demand for vaccines has increased. Current vaccine administration is mainly by injection. As mentioned above, since there are many pathogens involved in respiratory diseases, it is necessary to vaccinate several kinds of vaccines, which increases the number of injections and increases the stress on pigs. Two methods are conceivable as means for solving this problem. One is to change the method of administration to a method other than injection, and the other is to develop a combination vaccine effective against multiple diseases.

  Mycoplasma hyopneumoniae, one of the major virulence factors of swine complex respiratory disease, is a causative agent of swine mycoplasma pneumonia, and it has an economic loss because it affects pig gain and feed efficiency during the fattening period. Is big. It is also considered one of the first pathogens to trigger complex respiratory disease. Therefore, in Japan, vaccines are being controlled and the penetration rate is high. These vaccines are inactivated Mycoplasma hyopneumoniae cells or culture supernatants, with the addition of oil-based adjuvants or aluminum gel adjuvants as adjuvants. Inoculate once or twice.

  Swine erysipelas is a disease of pigs caused by infection with swine erysipelas (Ericiperos lichens) and has been occurring in pig farms around the world for over 100 years. The course is very acute and the fatality rate is high, and if the chronic course is taken, arthritis may be caused, and it may become a carrier pig. Since swine erysipelas is a zoonotic disease, if the test at the time of slaughter is positive, the individual will be abolished and the economic loss will be significant. Vaccines are effective in preventing swine erysipelas, and live vaccines made with attenuated fungi (Acuraflavin resistant strain: Koganei strain) are widely used in Japan. All vaccines used in Japan are administered by injection, but vaccines by oral administration are also commercially available in other countries.

  We have developed a 97 kilodalton protein with a molecular weight of 97 kilodaltons of the antigenic protein on the cell side, which is important for adherence to porcine tracheal cilia that work early in the infection of Mycoplasma hyopneumoniae. , The P97 adhesin gene is inserted into the gene of the infection defense-related protein SpaA.1 present on the surface of swine erysipelas, and the P97 adhesin protein is expressed on the surface of the swine erysozyme YS-19 Made. Furthermore, we confirmed that the porcine erysipelas YS-19 strain was administered intranasally to pigs and showed a good vaccine effect against the attack of swine venoms and Mycoplasma hyopneumoniae (see, for example, Patent Documents 1 to 3). reference.). We have succeeded in inventing the Y.S. vulgaris strain YS-19, which is a promising vaccine candidate strain that exerts vaccine effects on multiple diseases by administration methods other than injection. However, the route of administration of this strain was intranasal, and it was necessary to retain each individual during vaccine administration. This method of administration failed to meet the producer's request to reduce labor.

Currently, many types of swine mycoplasma pneumonia vaccines are commercially available from various manufacturers in Japan. As antigens for vaccines, those using mycoplasma cells are common, and some products using culture supernatant are seen, but all inactivated antigens are used. Adjuvants for the purpose of immunostimulation are generally added, and each product is devised as an adjuvant, but the administration method is intramuscular administration by injection, and systemic immunity is induced. However, local immunity cannot be induced.

There are two types of swine erysipelas vaccines, live vaccines and inactivated vaccines, both of which are highly evaluated for their effectiveness. The live vaccine in Japan uses the Koganei strain of swine erysipelas that has been managed as a seed lot for a long time, and its safety is high. Since the damage caused by swine erysipelas is primarily a sudden death due to bacteremia with systemic rhombus, the main component of the vaccine effect is systemic immunity.

In the pig farming industry, there is a tendency to refrain from the use of antibiotics. Instead, the use of vaccines is increasing for the control of swine diseases. Since pigs are exposed to many pathogens throughout their lives, they will be vaccinated as long as they cannot be eradicated. Vaccination is a great stress for pigs and sometimes affects weight gain. Therefore, the possibility of vaccination by oral administration was examined. In Japan, swine erysipelas live vaccine is administered by injection, but in the United States, oral administration by drinking water is carried out (see, for example, Patent Document 4). However, because the properties of the two vaccine strains are different, vaccines for oral administration are not commercially available in Japan. Although we examined the possibility of a vaccine for oral administration of the YS-19 strain that has already been developed, this strain was highly attenuated to enhance safety. The effect could not be imparted.

Japanese Patent No. 2992980 Japanese Patent No. 3793889 JP 2006-31824 A JP-T-2004-501979

Applied and Environmental Microbiology, January 1999, P278-282, Vol. 65, No. 1

  Accordingly, an object of the present invention is to provide a safe and effective vaccine that protects against the development of swine erysipelas and Mycoplasma hyopneumoniae by oral administration in order to reduce the stress on pigs caused by vaccine administration.

We have already cloned the gene of Surface Protective Antigen (SpaA.1), a swine erysipelas, inserted a foreign gene into this gene, created a chimeric gene, introduced the chimeric gene into swine gonococci, We have invented a porcine erysipelas that expresses. This time, we have further developed a method aimed at improving and stabilizing the efficiency of chimeric gene transfer and applied it to the development of an orally administered vaccine.
Usually, for transformation of Gram-positive bacteria, after cloning into a shuttle plasmid that can be replicated between Escherichia coli and the bacteria, it is introduced into the target strain and transformed. However, it is extremely difficult to genetically modify swine erysipelas for which no transformation method has been established.

The insertion of the p97 gene fragment onto the swine erysipelas chromosome introduces a chimeric gene designed by sandwiching the p97 gene fragment in the center of the spaA.1 gene of swine erysipelas by electroporation. Thereafter, it is carried out by homologous recombination between the spaA.1 gene on the chromosome and the spaA.1 gene on the plasmid. A case where the genetic recombination occurs in one place is called a single crossover, and a case where the genetic recombination occurs in two places at the same time is called a double crossover. When genetically modified bacteria are used as vaccines, they must be genetically stable. However, when a foreign gene is inserted in a single crossover, it is genetically unstable and drug resistance is selected. If cultured without pressure, that is, if antibiotics are not added to the medium, there is a possibility of returning to the original strain. In addition, since the entire gene on the plasmid including the antibiotic resistance gene remains on the chromosome, it is not suitable as a genetically modified vaccine. Therefore, the development of genetically engineered vaccines does not include antibiotic resistance genes or vector-derived gene parts by double crossover, and genetically stable recombination in which only the target gene (p97 gene) is introduced. The fungus must be made. However, after introducing a gene by transformation, the probability of homologous recombination occurring at a single crossover is very low, and furthermore, the probability of homologous recombination occurring at a double crossover is lower. Therefore, it is extremely difficult to select a clone in which homologous recombination has occurred in double crossover (see Non-Patent Document 1).

In the present invention, in order to increase the probability that homologous recombination will occur, an unknown gene region of about 1000 kb upstream and 700 kb downstream of the porcine Neisseria gonorrhoeae spaA.1 gene sandwiching the p97 gene fragment is newly cloned, A new recombinant plasmid was prepared and used in the experiment.
We confirmed the effectiveness of the oral administration of the porcine red venom Koganei strain, which has been proven as a live vaccine strain for injection for many years in Japan, as a host, and this was used as an attenuated vector for an orally administered mycoplasma vaccine. Selected as a porcine erysipelas. A part of the mycoplasma hyopneumoniae P97 adhesin gene was introduced into this bacterium by homologous recombination as described above, and a porcine erysipelas koganei strain expressing a part of the mycoplasma hyopneumoniae P97 adhesin was produced. When the vaccine effect of this strain was confirmed by an infection and onset prevention test in pigs, a good vaccine effect was confirmed against highly virulent swine erysipelas and virulent Mycoplasma hyopneumoniae.

  ADVANTAGE OF THE INVENTION According to this invention, the live vaccine which can be utilized as an infection protection vaccine of swine mycoplasma pneumonia and swine erysipelas is provided. The live vaccine can be produced in large quantities under the same conditions as normal swine erysipelas, and a vaccine comprising this as a component is highly safe and cheaper than a vaccine using whole mycoplasma cells. Become.

A gene sequence in which a part of Mycoplasma hyopneumoniae P97 adhesin was inserted into the gene of the porcine erysipelas Koganei strain. Continuation of Fig. 1 Shuttle plasmid construct

(Example 1) Construction of gene transfer plasmid Method of cloning both ends of SpaA.1: <BR> SpaA.1 is highly immunogenic. Therefore, phage clones that react with sera of swine erysipelas-infected super pigs are selected from the gene library of swine erysipelas, and the spaA.1 gene and the spaA.1 gene upstream and downstream regions are selected from them. The containing phagemid clone pER3 was selected. This is a clone in which an insert of about 4300 bp was inserted into a pBK-CMV vector (Stratagene). The EcoRI site fragment at the center of this gene was to be replaced with a fragment of the Mycoplasma P97 protein gene amplified by PCR so that the EcoRI site was added. Since there is an EcoRI cutting site on the side, it was found that the operation was not possible. Therefore, mutations were made in the form of removing the AGAATTCAAAAAGCTT sequence containing EcoRI on the vector side and the HindIII cleavage site, and a fragment of the Mycoplasma P97 protein gene amplified by PCR was inserted into the plasmid to add the EcoRI site. The entire sequence is shown in FIGS.

Plasmid constructs for transformation into swine erysipelas:
The insert having the above sequence was cleaved at the restriction enzyme site shown below, pGA14, which is a shuttle vector between Gram-positive bacteria and E. coli, and the p97 gene sandwiched between the spaA.1 genes was inserted. This was introduced into porcine erysipelas by electroporation, and a strain in which the upstream and downstream regions of the spaA.1 gene were replaced with the introduced gene by double crossover was selected.
The structure is shown in FIG.

(Example 2) Screening of foreign gene-transfected swine erysipelas For the purpose of confirming the expression of the mycoplasma antigen on the surface of the transformant obtained, the cultured cells were dot-blotted onto a nylon membrane, and anti-SpaA antibody and Anti-P97 antibody was sensitized and positive clones were selected. As a result, four positive clones ErMh-KoA, B, C and D were selected from the transformants of about 90 clones.

(Example 3) Screening using mice ErMhKo-A, B, C and D cultured bacterial solutions (10 ⁸ CFU / ml) were inoculated to 0.1 ml subcutaneously in the inner thigh of 4-week-old ddy mice. The presence or absence of life and arthritis was observed. Mouse of the LD ₁₀₀ of swine erysipelas bacteria strong Dokukabu is 10 ^3, the mouse will die if the toxicity return. The incidence of mouse arthritis was used as an indicator of clones that did not recover toxicity and retained immunogenicity.

  When the incidence of mouse arthritis in these clones was compared, the incidence differed from clone to clone (Table 1). Swine erysipelas ErMhKo-D showing the same incidence rate as the parent strain Koganei strain was selected for the pig test.

(Example 4) Vaccine Effect of Orally Administered Mycoplasma Vaccine 1) Effect on Swine Mycoplasma Pneumonia 11 SPF pigs of about 10 days are divided into 2 groups of 6 vaccine groups and 5 control groups, and the vaccine group contains ErMhKo. In the control group, the strain D was mixed with 10 ¹⁰ pig erysipelas Koganei strains per milk, each containing no antibiotics, and administered for 5 days. Two weeks after the final administration, a mixed solution of Mycoplasma hyopneumoniae and infected lung emulsion was intranasally administered for 3 consecutive days. Necropsy was performed 4 weeks after administration, and the rate of lung lesion formation was compared. As a result, there were no respiratory symptoms such as coughing or sneezing or fever in both groups during the breeding period after the attack. However, when comparing lung lesions in both groups at necropsy, as shown in Table 2, the rate of lung lesion formation in the vaccine group was significantly lower than that in the control group, and pigs orally administered with ErMh-D strains It was confirmed that the mycoplasma hyopneumoniae virulence strain shows a good lung lesion formation inhibitory effect.

2) Effects on swine erysipelas About 11 10-day-old SPF pigs are divided into two groups: five vaccine groups and two control groups. The vaccine group is ErMhKo-D strain and the control group is porcine erysentery koganei strain. Were mixed with 10 ¹⁰ milks per milk, each containing no antibiotics, and administered for 5 days. Two weeks after the final administration, highly virulent Fujisawa strain 5.0 × 10 ⁴ was administered intradermally. After the attack, the clinical symptoms and the state of rhomboid eruption were observed. As shown in Table 3, the control group disappeared from the day after the attack, and an increase in body temperature was observed. The rhomboid eruption metastasized throughout the body, pig No. 26 died on the 6th day, and pig No. 27 lay on the 6th day and was not euthanized. On the other hand, the vaccine group did not show clinical symptoms such as fever and loss of energy, and rhombus did not metastasize only at the site of inoculation, and a good vaccine effect was observed.

Indication for identification given by the depositor Receipt number EnMhKo-D FERM-AP-21759

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Claims (8)

  A porcine erysococcus having a portion of the Mycoplasma hyopneumoniae P97 adhesin protein expressed on the surface of attenuated porcine erysipelas having the ability to confer a vaccine effect by oral administration. 2. The swine erysipelas strain according to claim 1, wherein the attenuated swine erysipelas is a porcine red venom Koganei strain.   The swine erysipelas according to claim 1, wherein a foreign gene is inserted into the host DNA by double crossover. A vaccine using the strain according to claim 1. The vaccine according to claim 4, wherein living cells are used.   The vaccine according to claims 4 to 5, comprising a stabilizer and / or an adjuvant. A method for orally administering the vaccine according to claim 4 to induce immunity. A method for immunizing an animal by administering the vaccine according to claim 4 in drinking water or feed.