Method of obtaining toxin actinobacillus pleuropneumoniae apxi, applying culture medium, containing calcium-borogluconate complex. RU patent 2514667.

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

5 cl, 4 tbl

The invention relates to a method for RTX toxin ApxI by culturing bacteria Actinobacillus pleuropneumoniae in culture medium, which ensures the growth of bacteria, this environment, add calcium salt for education in the environment of calcium ions.

Pleuropneumonia pigs, the main respiratory disease of pigs, spread all over the world and causes heavy economic losses in pig production due to lightning deaths, ill pigs and delays in sales due to chronically infected animals. Etiological agent is Actinobacillus pleuropneumoniae. It is transmitted primarily through direct contact between animals, and received the infection leads to disease from lightning to the chronic. The disease, mainly, is an infection of the respiratory tract, with clinical signs of high fever, severe respiratory distress, cough and anorexia. Beginning of the disease quickly, morbidity and mortality are high. One of the ways of control of infection by bacteria Actinobacillus pleuropneumoniae (in the following also called "ARR") is a program of vaccination. In such programs have been used passivated bacteria, but aware of their heavy side effects. Currently widely used subunit vaccines based toxins RDAs.

RDAs produces the so-called RTX-toxins (RTX means repeat the toxin). The presence of these RTX-toxins making a great contribution to the pathogenic nature of this bacteria. RTX-toxins have been detailed previously described in the literature. As is well known, not all serotypes RDAs produce all RTX-toxins. For example, serotypes 1, 5, 9 and 11 produce ApxI and ApxII. Serotypes 2, 3, 4, 6 and 8 produce ApxII and ApxIII. Serotype 10 produces only ApxI, and serotypes 7 and 12 produce only ApxII. Currently commercially available vaccine against the RDA-based toxins ApxI, ApxII and ApxIII. Recently it was discovered that all serotypes RDAs produce fourth RTX-toxin, currently called ApxIV (see EP 0875574).

It is widely known how to produce RTX toxin-ApxI by cultivating Actinobacillus pleuropneumoniae in culture medium, which add salt of calcium (i.e. chemical compounds on the basis of the acid formed by replacing all or part of hydrogen ions acid on one or more of calcium ions). In particular, earlier in the EP 0453024 described this way (see "example 2", paragraph 2 "Purification and characterization of hemolysin", subsection "Methods"). Should take into account that used ApxI should be marked "HLY" (see Frey et al. in the magazine “J Gen Environ.”, August, 1993; 139(8): 1723-8). From this EP patent is known about adding on Wednesday calcium (CaCl2 ). In fact, article Environ Pathogenesis 37 (2004) 29-33 indicated that the transcriptional activity of the operon ApxI increases with the addition of growth in an environment of calcium. Thus, it can be ensured high levels of toxin ApxI. Environment must support the growth of bacteria RDAs. It is well known how to build an environment that ensures the growth of bacteria. Classical culture medium was originally developed Eagle, ham and other in 1950-60, They found that the environment that meets the needs of growth, must contain inorganic salts, nitrogen source (for example, in the form of nitrogen-containing compounds such as peptides or proteins)carbon source and vitamins. Environment mainly sautereau to prevent either from acidification; or alkalization. This basic recipe is available a large number of different compositions. For example, to provide the amino acids you can select the components of animal origin, but you can also choose chemically certain amino acids. In respect of other compounds also possible large number of variants. In fact, create an environment that ensures the growth of bacteria, is relatively easy. However, optimization of growth and/or receiving metabolites may take some time to develop, in particular, if the preferred environment that does not contain whey or other components of animal origin. Strategies to improve fermentation environment, however, well known in this area and described in detail in the literature (see, for example, a review article Kennedy and Krouse in journal of Industrial Microbiology & Biotechnology (1999) 23, 456-475). This optimization is part of the routine experiments in the laboratory fermentation. In the case of cultivation RDAs, NAD (nicotinamide adenine dinucleotide) essentially forms part of the environment because the bacterium RDAs is the NAD-dependent. In the absence NAD environment will not support the growth of bacteria Actinobacillus pleuropneumoniae and therefore can not be considered as a liquid environment to support the growth of the RDA from the point of view of this application and attached the claims. Environment to support the growth of bacteria or components to generate such environments commercially available in many firms, such as Sigma Aldrich, Quest International, Oxoid, Becton Dickinson, Pharmacia, VGD Inc, Mediatech, Invitrogen, Marcor, Irvin Scientific etc.

Although the ways of prior art is sufficient to produce economically meaningful output toxin ApxI, the applicant knew that there is a possibility of improvement. It is in the process of fermentation environment becomes cloudy. The merit of the applicants was the realization that this may be due to the precipitation of one (or several) of calcium salts. It RDAs produces carbon dioxide, which in the environment becomes carbonate ions. Calcium carbonate is a salt with very low water solubility. Because of this, you may experience some problems. First, suppose that the precipitation selects involved calcium ions, making them unavailable to bacteria RDAs. Secondly, precipitiously calcium salts cause problems related to processing. In particular, filters tend to become clogged. Therefore, the Complainant added on Wednesday, a large number of complexing agents to see if they can prevent the precipitation of salt. In fact, for example, adding EDTA environment can be more or less transparent. However, the use of such complexing agents affects getting ApxI. Therefore, the assumption seems to be incorrectly or incompletely. However, there is still a need to improve products ApxI.

Suddenly it was discovered that when using boroglukonata (for example, in the form of 2,3-dihydroxy-3-[2-hydroxy-5-(hydroxymethyl)-1,3,2-dioxaborolan-4-yl]propanoate; see also article Herbert Taylor MacPherson and James Stewart from the Institute of Moreton in Biochemical Journal: “Investigations on the nature of calcium borogluconate”, published on November 16, 1937) for education complex ions of calcium can be obtained ApxI at a high level in comparison with the methods of prior art that do not apply (not added) complexing agents or based on other complexing agents. Obviously, when using this specific complexing agent so that your environment has a complex calcium-borogluconate (for example, available in the form of D-gluconic acid, cyclic 4,5-ester with boric acid, calcium salt 2:1), can be prevented significant precipitation of calcium ions with other negative ions, at the same time, the calcium ions remain able to enhance the transcriptional activity of the operon ApxI bacteria Actinobacillus pleuropneumoniae. Apparently, calcium ions remain "trapped" in the complex of salt, where communications "takeover" on the one hand strong enough to prevent the formation of calcium ions precipitate, for example, carbonate or other negative ions, but on the other hand allow the bacteria to use calcium ions, if they are in free solution (i.e. form a complex with only water molecules). Apparently, borogluconate fully meets the critical balance that is necessary for the production ApxI bacteria RDAs.

In one of the options for the implementation of the concentration of boroglukonata is less than 60 mmol/L. Above this concentration is found that the products ApxI falls to low levels. Even if possible, it is preferable that the concentration remained below this figure. More preferably, concentrations were within the range from 25 to 45 mmol/l, in particular 40 mmol/l, which is optimum for some environments.

In another embodiment, calcium salt is borogluconate calcium. Although it is possible even, for example, the use of calcium chloride as a source of calcium and adding borogluconate salt for the formation of complex ions of calcium, it is preferable that calcium is supplied in the form borogluconate salt. Thus, there is no need to wait for a balance between a large number of physical reactions (precipitation, dissolution, destruction of the complex, formation of the complex)that occur in the environment. This saves time and therefore cost effective.

In another embodiment, in the process of cultivation through fluid leak air and air contains carbon dioxide above the atmospheric level. Suddenly it was shown that carbon dioxide increases the volume of production ApxI even more. It is noted, that basically is known about the use of the increased level of carbon dioxide during the cultivation of colonies of bacteria on cups (see, for example, U.S. patent 6019984: EXAMPLES "Bacterial strains and growth conditions"). However, this applies to the cultivation of colonies of bacteria, which are then used for inoculation of the fermenters. At this stage products RTX-toxins insignificant. More precisely, in General it is clear (see, for example, Microbial Pathogenesis 37 (2004) 29-33)that the maximum production Apx occurs when a high density of cells in fermenters, i.e. at the end of the exponential phase of growth. It is clear that at this stage the carbon dioxide is not suitable as a stimulating factor. Therefore, no attempt was made to increase production Apx through the use of increased levels of carbon dioxide. In particular, the Complainant discovered that the content in the air of 5% vol. carbon dioxide (net carbon dioxide to the volume of normal air) product ApxI is at very high level. It is noted that in this embodiment, to pass air through the environment can be used many techniques, usually with the help of a device that allows air bubbles to leak somewhere in the environment (i.e. under the surface of the environment). Under the "air" in the context of the present invention understand gaseous environment containing one or more of gaseous components that are normally present in the air, such as oxygen, nitrogen, carbon dioxide, helium, neon, argon, xenon, radon, etc.

The invention further additionally clarified with the following non-limiting examples.

MATERIALS AND METHODS

Bacterial strain and environment

The research was carried out using a strain of Actinobacillus pleuropneumoniae producing ApxI, serotype 10, later in this document called the RDA 10. In all cases, the working seed this strain was restored, using a Cup with a basis of Columbia blood agar (BAB) (production company Becton, Dickinson USA). Used liquid environment represented either Colombian culture medium (production company Becton, Dickinson USA)supported the pH value of 7.3 using NaOH and acetic acid or the environment, contains no components of animal origin (called "environment ACF"). Last Wednesday contains MgSO 4 (0.75 g/l), cysteine·HCl (0.1 g/l), FeCl 3 (0.1 g/l), NaNO 3 (0.1 g/l), KCl (0.1 g/l), trace elements (such as a 2.5 ml solution SL-10, specified in the manual Handbook of Microbiological Media, 3 rd rdition, Ronald Atlas, CRC press, 2004), a 50% glucose solution (10 ml) and 10 mm the solution of amino acids (contains all 20 amino acids, with the exception of tryptophan), HEPES buffer (6 g/l; for example, available from the company Sigma Aldrich) and yeast extract (10 g/l; for example, available from Becton, Dickinson).

These have been used in rekultivierung and fermentation. Nicotinamide adenine dinucleotide (0,01%) and calcium (various concentrations) was used preculture and fermentation. All environment sterilized filtering with pore diameter of 0,22 microns. Before using fermentation environment was heated at 85 OC for one minute.

Cultivation

Working seed strain RDAs 10 were sown for a Cup of Colombian agar VAV and incubated for about 24 hours at 37 deg C. Several colonies were selected for inoculation of the vessel with a volume of 500 ml, containing 75 ml Columbia nutrient medium. The vessel is incubated for about 6 hours at 37 C with shaking for education preculture. Through this preculture carried out several fermenteze. Some of them carried in containers with a volume of 500 ml, In this case 75 ml environment was inocularea 1 ml of preculture. The vessels were incubated at 37 C with shaking. Alternative cultivation was carried out in fermenters SIXFORS (the company's products Infors AG, Switzerland), containing approximately 400 ml of culture medium, which added 20 ml preculture as inoculum. The temperature of cultivation is also 37 deg C.

Analyses

Cell growth was determined by measuring the optical density (OD) at 660 nm. The concentration of antigen ApxI was measured using the enacted ELISA.

The first experiment was conducted to determine whether more calcium, despite the formation of complexes with boroglukonata, for bacteria RDAs. This experiment was carried out in containers, as described in this document above. The results are presented below in table 1.

As indicated by the data of table 1, in the formation of complex ions of calcium boroglukonata can be obtained good output ApxI. An important advantage of education of this complex is that the precipitation of calcium salts is no longer a significant impact on the course of the process.

The second experiment was carried out to understand the effects of borogluconate in an environment that contains no components of animal origin. To do this, the authors compared the addition of 20 mm solution CaCl2 with the addition of 20 mm mortar Sa boroglukonata. The results are presented in table 2.

Wednesday

Antigen ApxI (U/ml)

ACF added 20 mm CaCl2

ACF added 20 mm Sa-borogluconate

Received two results. First, it is clear that the use of calcium chloride obtaining sufficient quantities ApxI in the environment ACF difficult even with the establishment of normal levels of calcium. In the formation of complex calcium boroglukonata can be obtained high output ApxI. Comparable results can be obtained in other operating environments. The authors have carried out such an experiment in an environment that did not include any of ferric chloride or of magnesium sulfate ("ACF-alt"), but otherwise represented the same environment as the environment ACF described in this document above. And this time in the formation of complex calcium boroglukonata received increased substantially levels.

The third experiment was carried out to study the effect of concentration of boroglukonata. The authors used three different concentrations, namely 20, 40 and 60 mm boroglukonata calcium. The results are presented in table 3.

Wednesday

Antigen ApxI (U/ml)

ACF added 20 mm Sa-borogluconate

ACF, added 40 mm Sa-borogluconate

ACF added 60 mm Sa-borogluconate

As is clear from table 3, the optimal concentration of about 40 mm.

In the fourth experiment, the authors studied the influence of increased levels of carbon dioxide to receive ApxI. To do this, the authors used the environment ACF-alt described herein above, and increased the level of sodium nitrate up to 0.5 g/L. the Concentration of boroglukonata varied between 40, 50 and 70 mm. The increased concentration of CO 2 received, maintaining a constant flow of air in the fermenter 1 vvm (volume of gas in the volume of the environment per minute) for a mixture of air/2 95/5 about./about. The experiments were carried out in the fermenter SIXFORS, as described in this document above. The results are presented in table 4.

Wednesday

Antigen ApxI (U/ml) ELISA

ACF-alt added 40 mm Sa-borogluconate, 5% WITH 2

ACF-alt added 50 mm Sa-borogluconate, 5% WITH 2

ACF-alt added 70 mm Sa-borogluconate, 5% WITH 2

Based on these results, we can conclude that carbon dioxide has a positive effect on production ApxI: even at a concentration of 70 mm boroglukonata calcium can be obtained acceptable levels ApxI. And in this case, the optimal concentration of 40 mm.

CONCLUSION

The Complainant discovered that liquid culture medium that supports the growth of bacteria RDAs, borogluconate can provide the ultimate balance between prevent precipitation of calcium ions with negatively charged ions, on the one hand, and the maintenance of calcium ions available to stimulate Actinobacillus pleuropneumoniae to products ApxI, on the other hand. It can be used mainly in any medium for cultivation of Actinobacillus pleuropneumoniae containing negative ions forming the precipitate calcium ions. In fact, depending on your environment and optimization of its components bacteria RDAs will produce a higher or lower levels ApxI. But because of the shielding effect of boroglukonata will work regardless of the actual rate of production of bacteria, this solution can be successfully used for all environments, in particular because, in fact, all environments contain carbonate ions, which are ions that can form a precipitate calcium ions.

1. The method of obtaining RTX toxin ApxI by culturing bacteria Actinobacillus pleuropneumoniae in a culture medium, which ensures the growth of bacteria, and to this environment, add calcium salt for education in the environment of calcium ions, wherein the cultural environment includes borogluconate at a concentration of less than 60 mmol/l for education in the environment of complex calcium-borogluconate.

2. The method according to claim 1, characterized in that the concentration of boroglukonata is in the range from 25 to 45 mmol/l, preferably 40 mmol/L.

3. The method according to claim 1 or 2, wherein the calcium salt is borogluconate calcium.

4. The method according to claim 1, characterized in that in the process of cultivation through fluid leak air and air contains carbon dioxide above the atmospheric level.

5. The method according to claim 4, wherein the air contains 5% vol. of carbon dioxide.