JP5913450B2 - Orally administered immunostimulant for aquaculture - Google Patents

Description

  The present invention relates to an orally administrable immunostimulant for aquaculture.

  Aquaculture appears to be the only way to cover the growth requirements of aquatic foods that cannot be covered by harvested fisheries. World aquaculture production has increased from less than 1 million tons in the early 1950s to 59.4 million tons by 2004 over the past 50 years. This production level is worth US $ 70.3 billion in US dollars. 69.6% of global production is produced by China, 21.9% by other Asian and Pacific countries, 3.9% by Western European countries, and 0.4% by Central and Eastern European countries. It was. In relation to the environment, the amount of aquaculture produced by underwater aquaculture (seawater aquaculture) in fiscal 2004 was 30.2 million tons, which corresponds to 50.9% of the total aquaculture. Aquaculture with fresh water is 25.8 million tons (43.4%) and the rest is 3.4 million tons (5.7%), which are derived from production in brackish environments. All data listed here are based on the Fisheries Technical Paper 2006, State of World Aquaculture (FAO).

  European aquaculture production is only 3% of global production. However, several species such as Atlantic salmon, trout, sea bass, sea bream (gilthead sesbream), turbot (flat fish such as carp or sea bream, eg sand flounder), and mussels It is the world's top production volume. The production volume in 2002 was over 1.3 million tons and worth € 3,280 million. The most important species in the Mediterranean countries are European sea bream, seabass and turbot. In 2002, the production of European red sea bream and seabass was 181,000 tons, and the main producers were Greece, Turkey, Spain, Italy and France. In 2002, turbot production was 5320 tons, 75% of which was produced in Spain. These data are all based on FAO data.

  Fish diseases hinder the development of aquaculture and cause severe blows to economic development in many countries around the world. Diseases often cause massive death and cause significant production declines. Loss of aquaculture also causes reduced food availability, loss of income and employment, all of which are accompanied by incidental social significance. The most common diseases that attack the aforementioned species and the pathogens (bacteria or viruses) that cause them are Vibrio (Photobacterium damselae subsp. Damselae, Vibrio spp.), Pasteurella (Photobacterium damselae subp. Pasteurella), Luminescent bacilli (Photobacterium damselae subp. Pasteurella.), Tenacibaculum maritimum, Myxomycosis (Flexibacter maritimus), Atypical eromonas (Aeromonas salmonicida), Streptococcus infection (Streptococcus parauberis), Winter deficiency syndrome (Pseudomonas anguilliseptica) , Viral neuronecrosis (Nodavirus), lymphocystis (Iridoviridae), dilated bowel syndrome (virus-like particles), infectious pancreatic necrosis (IPNV), infectious hematopoietic necrosis (IHNV) or viral hemorrhagic sepsis ( VHS). All these data are based on the Cultured Aquatic Species Information program (FAO). The aquaculture state of the species in the aquaculture farm (i.e. high-density aquaculture fish or aquaculture environment, etc.) is suitable for infectious disease pathogens transmitted between solids. In addition, the stress experienced by fish farmed in fish farms will cause a reduction in the immune system that facilitates infection by pathogens. Thus, much research effort has been devoted to the development of products such as vaccines or immunostimulants to prevent fish diseases. Disease prevention also reduces environmental impact. This is because large amounts of antibiotics are avoided in aquaculture. Several products have been used to vaccinate fish or activate the immune system. In fact, normal bacterial fish diseases such as vibriosis, gonococcal luminosis, atypical eromonosis, gliding bacteriosis, winter insufficiency syndrome or streptococcal infection etc. (reported by Toranzo et al., 2005 and Sommerset et al., 2005) There are vaccines that can be used for normal viral fish diseases such as IPNV or IHNV.

  There are three general methods for vaccination consisting of immersion, injection and oral administration. Intraperitoneal injection is the most effective method for vaccination. This is because intraperitoneal injection allows the dose to be freely controlled, and an adjuvant that provides an excellent immune response can be used. However, the intraperitoneal injection method has drawbacks. For example, sedation and injection operations on fish can stress the fish and damage the fish if the vaccine is not administered with great care. Furthermore, small fish cannot be vaccinated by this method. Immersion methods are frequently used in farms. Because it is simple and quick. However, it is too stressful for fish with inconsistent dose control and consistent immune system suppression. The oral administration of the vaccine does not require fish manipulation, and is a method suitable for vaccination of fish of all sizes, including young fish that have just started feeding. This method of vaccination of young fish is extremely important. This is because these juveniles are very susceptible to pathogens. Oral administration also has some drawbacks. For example, the dosage cannot be strictly controlled, and a large amount of antigen is required for immunization. The most important obstacle to oral administration is that the antigen is often inactivated in the intestine by acidic environment or protease activity, resulting in inhibition of active antigen absorption by the intestine. For this reason, it is necessary to protect the antigen for effective oral administration. That is, it is necessary to encapsulate the antigen to avoid intestinal antigen degradation (Hart et al., 1988; Quentel and Vigneulle, 1997). Alternative methods for protecting antigens from degradation (eg, confinement within liposomes or alginate microencapsulation) illustrate some promising results (Ire et al., 2005; Maurice et al., 2004) .

  Immunostimulants are used in aquaculture to increase fish disease resistance. Many different types of immunostimulants are known, depending on their chemical properties and mode of action. For example, various β-1,3 glucan products derived from bacteria and mycelium, β-1,3 / 1,6 glucan derived from yeast cell walls, and composites derived from various biological sources, depending on the structural elements of bacteria (LPS) Peptides, nucleotides, synthetic products (levamisole) and vitamins present in carbohydrate structural structures (glycans), extracts (extracts) produced by enzymatic hydrolysis of certain animal or fish proteins C (researched by Raa, 1996). Also, some immunostimulants used enhance specific antibody responses (studied by Sakai, 1999). Several conditions are suitable for use of immunostimulants in aquaculture. For example, fish manipulation, water temperature changes, high concentration pathogen exposure, vaccine adjuvants and all high stress situations. Numerous publications have been published regarding the effects of immunostimulants in the European sea bream (gilthead sesbream) immune system. For example, Ortuno et al. 2002 and Rodriguez et al 2003 paper on budding yeast (Saccharomyces cerevisiae), Esteban et al 2002 paper on chitin, Esteban et al 2001 paper and Cuesta et al 2003 paper. Papers, Cuesta et al 2002 paper on vitamins C and E, Ortuno et al 2003 paper and Mulero et al 1998 paper on levamisole and Cuesta et al 2002 paper. Of the immunostimulants known to be effective in fish, glucan, chitin and levamisole enhance phagocytic activity, while yeast glucan and vitamin C activate complement activity. Several studies on the administration of B-1,3 / 1,6 glucans as immunostimulants have shown excellent results with regard to improving the survival rate of halibut larvae in critical aspects of the developmental stage of halibut larvae. Is presented in a 1999 paper by Ottesn, Lunda and Engstad et al., And increased resistance to bacterial infections is published in a 1990 paper by Raa et al. And Robertsen et al. In a 1990 paper. Increased effectiveness is published in Raa et al.'S 1990 paper, Rorstad, Assjord, and Robertsen's 1993 paper.

Cytokine regulates the amount of heavy biological processes such as cell growth, cell activity, inflammation, immunity, tissue repair, fibrosis and morphogenesis, so the general definition of “immunomodulator” Proteins belonging to Cytokines are different groups of proteins that share several properties and are essential for the development and action of both mediator effector phases of the innate and adaptive immune responses. Over the past few years, a large number of cytokines have been cloned and sequenced in several fish genera (see, for example, Bird et al. 2002). Cytokines can be classified into the following four groups according to their functions.
(A) Cytokines that mediate innate immunity. The cytokines with the best properties in this group of fish are type I interferons (IFN), which include IFNα and IFNβ, the interleukin 1 (IL-1) family (IL-1α, IL-1β, IL-1 receptor Antagonists and IL-18) and tumor necrosis factor (TNF) family (including TNFα, TNFβ, Ltβ and Fas ligand). Of these, TNFα is the most important member with immune system regulatory functions.
(b) cytokines that regulate hematopoiesis,
(c) cytokines that regulate lymphocytes and
(d) Cytokines that control non-specific effector cells.

  In these groups, the cytokine with the most excellent properties is interleukin 2 (IL-2) in the case of group (b) and transforming growth factor beta (TGFβ) in group (c).

  TNFα is an inflammatory cytokine produced by monosite / macrophages during acute inflammation and is responsible for responding to precursor events in different areas within the cell. TNFα serves as an important mediator in resistance to parasitic, bacterial and viral infections (Czarniecki's 1993 paper, Goldfeld and Tsai's 1996 paper, and Steinshamn et al.'S 1996 paper. reference). TNFα also has other important therapeutic effects. For example, tumor resistance (see Vilcek and Lee 1991 paper), sleep regulation (see Krueger et al. 1998 paper) and embryo growth (see Wride and Sanders 1995 paper). TNFα exerts many of these actions by binding as a trimer to the cell membrane receptor, TNFR-1 or TNFR-2 (see Dembic et al. 1990 and Loetscher et al. 1990). Both of these receptors are present in very large amounts in most cells (see Loetscher et al. 1991 and Schoenfeld et al. 1991).

  TNFα mRNA appears to be transcribed in a wide variety of cells. Therefore, it is remarkably suppressed at the post-transcriptional level (see Han et al. 1990 paper). TNFα exists in two forms, a membrane bound form and a soluble form. Each form probably has a distinct physiological role (see Watts et al. 1990 paper). TNFα is manifested as a 26 kDa membrane-bound precursor, which is proteolytically cleaved by disintegrin and metalloproteinase (TACE: TNFα converting enzyme) to produce a 17 kDa C-terminal active form (Black et al. 1997) And the 1997 paper by Moss et al.).

  Some mammalian TNFα information has been fully or partially sequenced. Such mammals include, for example, humans (see Pnnica et al. 1984 paper), chimpanzees (see Kutsui et al. 2002 paper), mice (see Shirai et al. 1988 paper), dogs (Zucker et al. 1994 Or cat (see McGraw et al.'S 1990 paper). Some fish TNFα information or TNFα information fragments have also been sequenced. Such fish can be black seabream (see Cai et al. 2003), rainbow trout (see Laing et al. 2001), carp (see Saeij et al. 2003) or zebrafish (Phelan et al.). (See 2003 paper) or European sea bream (see Garcia-Castillo et al. 2002 paper).

  The European red sea bream TNFα information has 4 exons, 3 introns and a length of 1244 pb. The cDNA consists of 1359 pb, which contains an open reading frame (ORF) of 762 pb, a 5 'untranslated region (UTR) of 142 pb and a 3' UTR of 455 pb. The putative protein has 253 amino acids, has an approximate molecular weight of 28 kDa, and provides high sequence homology with the other fish species TNFα, particularly in the C-terminal extreme required for interaction with the receptor.

  The TNFα protein includes a transmembrane region between residues 37 and 54 and the conserved sequence Thr-Leu associated with TACE protein cleavage (see Garcia-Castillo et al. 2002 paper). Therefore, a 253 amino acid membrane bound protein (proTNFα) and a 167 amino acid soluble protein (sbTNFα) can be distinguished.

  Expression studies revealed constitutive expression of TNFα in all tissues of the European sea bream tested such as liver, gill, blood, head kidney, peritoneal exudate, brain and spleen. This is a macrophage that is one of the main sources of this molecule.

  Patent Document 1 (US Pat. No. 5,871,751) discloses a vaccine for treating fish and a method for treating fish that are susceptible to infection by fish bacterial kidney disease (Renibacterium salmoninarum). This vaccine consists of killed microorganisms that lack the intact cell surface binding protein p57 and is provided with an enteric coating for oral administration. Enteric solvent skin does not accept dissolution or degradation in the stomach, but consists of a polymer coating that dissolves when it passes through the high pH environment in the intestine. A preferred embodiment of the disclosed vaccine is coated with a first layer of dead microorganisms produced using spherical sugar microspheres and lacking the intact cell surface binding protein p57, and then lysed in the stomach of fish. It is covered with a second layer of enteric coating made of a material that does not accept decomposition.

  Patent Document 2 (International Publication No. WO03 / 020040A1) discloses a vaccine comprising a multicellular organism encapsulating a single cell microorganism containing an antigen. This vaccine is used to feed aquatic animals (eg fish) to be vaccinated. According to Patent Document 2, antigens expressed in unicellular microorganisms are delivered to aquatic animals by two-stage feeding. That is, a first stage in which a unicellular microorganism (containing an antigen) is fed to a multicellular organism and a second stage in which the multicellular organism is fed to an aquatic animal. According to Patent Document 2, the simultaneous presence of both unicellular microorganisms and multicellular organisms is always necessary. The antigen to be expressed depends on whether the elicited immune response is against the target pathogen. Patent Document 2 discloses Pseudomonas aeruginosa exotoxin A (PE) as a bacterial antigen, E. coli as a unicellular microorganism, and Artemia nauplii as a multicellular organism. Since US Pat. No. 6,057,086 discloses the production of an orally administered vaccine, the selected antigen depends strictly on the target pathogen to which this vaccine must be directed.

  Patent Document 3 (Spanish Patent Publication No. 2189508A1) discloses a method for producing 1L-1β recombinant of European red sea bream (Sparus aurata L.), a vaccine adjuvant, and a method for use as an immunostimulant in commercial fish. Yes. The production of the 1L-1β recombinant according to Patent Document 3 is carried out by cloning it into an E. coli expression vector. The recombinant protein thus obtained is purified and used as an immunostimulant and vaccine adjuvant in fish mass culture.

US Pat. No. 5,871,751 International Publication No. WO03 / 020040A1 Spanish Patent Publication No. 2189508A1

  An object of the present invention is to provide an immunostimulant suitable for oral administration that is not subject to hydrolysis or degradation and is completely absorbed by the microorganism to be administered.

  Another object of the present invention is to provide an immunostimulant that can be administered directly to a target subject.

  Another object of the present invention is to provide an immunostimulant that can be absorbed by microorganisms administered in large amounts relative to the initial dose.

  Yet another object of the present invention is to provide an immunostimulant that is not specifically delivered to a specific pathogen or a specific group of pathogens.

  Still another object of the present invention is to provide a method for producing an orally administrable immunostimulant and a method for using the immunostimulant for activating an immune response in aquaculture.

  In order to solve the above-mentioned problems, the present invention provides at least a microencapsulated recombinant fish cytokine, in particular, an orally administrable immunostimulant consisting of tumor necrosis factor α (TNFα) and a microencapsulated recombinant fish cytokine. A method for producing an orally administrable immunostimulant comprising the following and its use for activation of immune response in aquaculture.

  The immunostimulant of the present invention is characterized in that the active molecule acting as an immunostimulant (ie, cytokine and specific tumor necrosis factor α (TNFα)) is a cytokine belonging to the same fish species for which activation of the immune response is desired. That is. This feature is very important. This is because the immune response of a specific fish species that positively enhances a specific set of immunostimulants already belonging to that fish species can be activated. This fact means the following: That is, there are no external factors, compounds or products that are administered to the fish species by any method, and as a result, the results obtained are not only excellent in profit but also excellent as a safe approach. As described above, according to the present invention, the active molecule acting as an immunostimulant is a cytokine obtained from fish itself, tumor necrosis factor α (TNFα). Accordingly, when the immunostimulant of the present invention is administered, no foreign substance is introduced into the fish. As a result, the possibility of side effects is avoided.

  According to the present invention, a gene encoding a specific cytokine is isolated from fish tissue and cloned into an expression vector for cytokine expression in a suitable host microorganism. Host cells are cultured in bioreactors using solvents suitable for optimal cytokine expression and high biomass production. According to a preferred embodiment of the present invention, the host microorganism is yeast (yeast), preferably methanol-utilizing yeast (Pichia pastoris) and budding yeast (Saccharomyces cerevisiae), most preferably methanol-assimilating yeast. The selection of yeast as the host microorganism is not a coincidence. This is because yeast is a eukaryotic microorganism and can express specific cytokines both outside and inside the cell. This makes it possible to determine the recombinant protein expression method that has many effects on the fermentation method and the purification of the product thus obtained. Then, the recombinant protein (either obtained alone or obtained inside the host microorganism) is microencapsulated, and if necessary, through a purification treatment step, an administrable microencapsulated protein is obtained. can get. As used herein, the term “microencapsulation method” refers to anything suitable for applying at least partially a coating to a recombinant cytokine, which coating is suitable to protect the cytokine from intestinal degradation. Means.

  In fact, when an immunostimulant is administered orally, an efficient protection method is necessary to avoid intestinal degradation of the immunostimulant and to allow complete absorption by the intestinal tract. Microencapsulation is a technical method of obtaining microparticles by enclosing or coating very small liquid or solid droplets or particles with a polymeric material. Microencapsulation technology is widely used in several biological and chemical systems and can produce vast quantities of products. According to the present invention, the primary technique used to successfully obtain microencapsulated formulations of the desired and specific cytokines (particularly TNFα) is micronization by spray drying. In the technique of atomization by spray drying, the active molecules are dissolved or suspended in a molten or polymer solution and are trapped in dry particles. According to the present invention, the most useful polymers used to protect recombinant protein microparticles from intestinal production pH are, for example, cellulose acetate phthalate, hydroxypropylcellulose phthalate, carboxymethylcellulose, methacrylic acid copolymers ( For example, methacrylic acid copolymer LS) such as Eudragit L-30 and Lollicoat is selected. Other polymers such as polymers selected from maltodextrin, chitosan, gelatin, starch or gum arabic can also be used.

  In the present invention, TNFα is used as an example of a cytokine that is expressed in microorganisms and then mixed with a polymer to protect the protein from the acidic environment and protease activity in the gut. The recombinant protein, ie TNFα, can be used as it is when the host microorganism allows its overexpression outside the cell. As an effective alternative, when the host microorganism overexpresses the protein internally, the same microorganism can be microencapsulated to obtain a microencapsulated microorganism that can be used as it is, and all the effects as described above can be obtained. Demonstrated. The overexpressed protein is dried by spray drying and added to the fish feed in an amount effective for fish health. The product according to the invention has tremendous importance in the aquaculture market to prevent fish diseases by infection, protect fish in some high stress environment and as an adjuvant for vaccines.

  According to the present invention, when the immunostimulant is directed to the treatment of adult fish, the immunostimulant can always be administered as it is because of the advantages obtained by the microencapsulation process. In contrast, if the immunostimulant is directed to the treatment of juveniles or juveniles, for example, feeding the immunostimulant to a multicellular organism such as Artemia nauplii, then larvae or This multicellular organism, which can be easily fed by the fry school, can be fed to young or fry. In either case, the microencapsulated immunostimulant can safely maintain the main properties and activity of the product and provides great benefits for aquaculture.

  As an example, recombinant sbTNFα of European sea bream was purified and its biological activity was evaluated in vivo and in vitro. The recombinant protein contains 6 histidines at the N-terminal extreme for detection of anti-polyhistidine mAbs in affinity chromatography purification and immunoblotting. Intraperitoneal injections consist of (a) priming of peritoneal effusion and respiratory burst of head kidney (HK) leukocytes, (b) rapid solid increase of phagocyte granule leukocytes to the injection site and (c) in HK. Induction of granulocyte formation occurred. sbTNFα was able to induce strong shoot proliferation of HK cells in vitro.

  Another advantage of the present invention is that the host microorganism expressing the TNFα cytokine itself can function as an additional source of immunostimulatory material. Some structural elements of bacteria, glucans from the cell walls of yeast or complex carbohydrate structures (glycans) from various biological sources can in fact be used as immunostimulants.

  The invention is explained in more detail by the following examples, which are carried out using TNFα of several fish species of great importance in aquaculture. However, other fish species TNFα and other cytokines can also be used to practice the invention. The present invention can also be applied to cytokines extracted or generated by synthesis.

The primers used for gene construction are shown. EcoRi and BamHi restriction sites for cloning are shown in italics. A DNA fragment containing sbTNFα of European sea bream and a deduced protein sequence are shown. The DNA fragment containing the European sea bream His ₆ -sbTNFα and the deduced protein sequence for expression in methanol-utilizing yeast. The underlined portion in the figure shows 6 CAT / C codons and histidines. The consensus sequence for protein expression in methanol-utilizing yeast is highlighted. EcoRi and BamHi restriction sites are shown in italics. The DNA fragment containing the sea bass His ₆ -sbTNFα and the deduced protein sequence for expression in methanol-utilizing yeast are shown. The underlined portion in the figure shows 6 CAT / C codons and histidines. The consensus sequence for protein expression in methanol-utilizing yeast is highlighted. EcoRi and BamHi restriction sites are shown in italics. The DNA fragment containing turbot His ₆ -sbTNFα and the deduced protein sequence for expression in methanol-utilizing yeast are shown. The underlined portion in the figure shows 6 CAT / C codons and histidines. The consensus sequence for protein expression in methanol-utilizing yeast is highlighted. EcoRi and BamHi restriction sites are shown in italics. A shows the result of electrophoresis by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and B shows the result of Western blot analysis by anti-polyhistidine mAb. The arrows in the figure indicate His ₆ -sbTNFα of European sea bream (Sparus aurata) expressed in (1) E. coli, (2) budding yeast, and (3) methanol-utilizing yeast. It is a microscopic observation figure of Artemia nauplii larvae (Artemia nauplii) after feeding the stained recombinant yeast for 60 minutes. Show. It is a graph which shows the respiratory burst activity by the luminol dependence chemiluminescence produced | generated by the head kidney leukocyte. The vertical axis represents chemiluminescence (relative light units / minute) Figure 2 is a graph showing mRNA levels of several inflammatory genes in the head kidney and intestinal tract. The upper graph relates to the intestine and the lower graph relates to the head kidney. Both vertical axes represent the rate of increase relative to the control.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Expression of European red sea bream TNF.ALPHA. in Escherichia coli cloning sbTNF.ALPHA. into pET15b
LPS-stimulated head kidney cDNA was used as a template in PCR for amplification of sbTNFα by FE4 and RE5 (see FIG. 1). Both primers have a BamHI restriction site for post cloning of the PCR product at the same site of plasmid pET15b (Novagen). Amplification was performed in a sample containing a cDNA template, each dNTP 50 μM, primer 0.2 mM, 1 × buffer PLUS containing MgCl ₂ and 1 unit of Eco Taq PULS DNA polymerase (manufactured by Ecogen). The cycle reaction was performed in an Eppendorf Mastercycler Gradient at 95 ° C for 2 minutes for 1 cycle, 95 ° C for 45 seconds for 25 cycles, 60 ° C for 45 seconds for 25 cycles and 72 ° C for 30 seconds for 25 cycles followed by 72 A cycle of 10 ° C. for 10 minutes was performed. The PCR product was purified with the QIAquick PCR purification kit (Qiagen), using 1 unit of T4 DNA ligase (New England Biolabs), and plasmid pGEM-T Easy (3: 1) in the relationship of insert: plasmid (3: 1). Promega) and room temperature for 16 hours. The ligation mixture was used against transformed E. coli DH5α competent cells (transformed recipient cells) and spread into LB plates containing ampicillin and X-Gal (Sigma). The plate was incubated at 37 ° C. and some resulting white colonies were picked and tested for the presence of the insert through plasmid isolation with the QIAprep · Spin · Miniprep kit (Qiagen) and digestion with BamHI. A particular plasmid containing the insert (sbTNFα with BamHI ends, see FIG. 2) was designated pVP81. This plasmid and 500 ng of pET15b were digested with 10 units of BamHI for pVP81 sbTNFα release and for pET15b linearization. Both the insert and linear plasmid were separated by electrophoresis in an agarose low melting gel (Pronadisa) with the QIAquick gel extraction kit (Qiagen), purified, and 1 unit of T4 DNA ligase (New England Biolabs) and ligated for 16 hours at room temperature. The ligation mixture was used against transformed E. coli DH5α competent cells and spread into LB plates containing ampicillin. The plates were incubated at 37 ° C. and several productive colony plasmids were isolated and tested for insert release by BamHI digestion and insert orientation by Pvull digestion. The sequence of the selected plasmid was determined with an ABI Prism377 gene analyzer (CIB, manufactured by CSIC).

Transformation of E. coli and measurement of expression Escherichia coli BL21 (DE3) competent cells were transformed with sbTNFα-containing plasmid pET15b, and the resulting mixture was spread in ampicillin-containing LB plates. Several productive colonies were cultured overnight in LB-ampicillin medium. After dilution in fresh LB-ampicillin, the culture was grown to OD ₆₀₀ = 0.8 at 37 ° C. and 0.25-4 using 1 mM isopropyl-D-thiogalactoside (IPTG; Applichem). Induced at 25 ° C or 37 ° C for hours. The protein expression in the whole cell extract was checked by centrifuging (1400 rpm) 0.1 ml of induction culture and the cell pellet was Western blotted using SDS-PAGE and anti-polyhistidine mAb (Sigma). For analysis by HPLC, it was dissolved by boiling in SDS-loading buffer solution.

Expression of European sea bream sbTNFα in budding yeast
Using TNF-ECOF and TNF-ECOR primers sbTNFα containing plasmid pET15b as the template in PCR with (see Figure 1) for amplification of the fragment containing the His ₆ -sbTNFα cloning 613pb to P424-GPD of His ₆ -sbTNFα . Both primers contain an EcoRI restriction site for cloning the PCR product within the same site of plasmid p424-GPD (ATCC87357). Amplification template 5 ng, the MgCl ₂ 2.5 mM, each dNTPs 50 [mu] M, the primers 0.2mM, 1X · High · Speec additive, a 1X buffer PLUS and Eco · Taq · PLUS · DNA polymerase containing 2 units Performed in the sample. Cycle reaction in Smart Cycler II (Cepheid) 1 cycle at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 30 seconds, 30 cycles at 62 ° C for 45 seconds and 30 cycles at 72 ° C for 2 minutes, Subsequently, 72 ° C. and 10 minutes were performed in the order of one cycle. PCR products were separated by electrophoresis in 0.8% agarose gel (Pronadisa) containing 0.5 μg / ml ethidium bromide and purified with QIAquick gel extraction kit (Qiagen). The purified fragment and 250 ng of p424-GPD were digested with 10 units of EcoRI (Fermentas) at 37 ° C. and then ligated at 22 ° C. with 5 units of T4 DNA ligase (Fermentas). The ligation reaction product was transformed into E. coli DH5α competent cells and plasmid-containing colonies were selected in LB plates with ampicillin 100 μg / ml. Recombinants were identified by colony PCR using 2X • PCR • Master • Mix (manufactured by Fermentas) and PRO-GPD and TER-GPD primers (see FIG. 1). The resulting His ₆ -sbTNFα-containing plasmid p424-GPD was purified with the QIAprep / Spin / Miniprep kit (Qiagen) and tested for insert release after digestion with EcoRI and insert orientation with digested Smal and Ncol. Moreover, the sequence of the plasmid was determined by ABI Prism 3130 gene analyzer (SACE, University of Murcia).

Transformation of budding yeast and measurement of expression The budding cell strain used for transformation was EGY48 (Invitrogen). To make EGY48 for transformation, 100 ml of a culture grown in OD P medium (2% peptone, 1% yeast extract, 2% glucose) to OD ₆₀₀ = 0.5 at 2500 rpm at 4 ° C. Centrifuge for 5 minutes. After washing with sterilized water, the cells were resuspended in 1X · LiAc-TE (0.1 M lithium acetate, 10 mM Tris pH 7.5, 1 mM EDTA pH 8.0). For transformation, 10 μl of DNA at a concentration of 0.3 μg / μl was mixed with 50 μl of cells and 50 μg of DNA carrier (used after boiling salmon DNA in advance). Subsequently, this mixture is added to the 1X · LiAc-TE-PEG (1X · LiAc-TE is the same as above, PEG is 40%) mixture and incubated at 30 ° C for 30 minutes, after which DMSO is added. Added to 10% and incubated at 42 ° C. for 10 minutes. Cells are spread on MMSS + SDC-Trp (minimum medium containing 2% glucose, 0.17% yeast nitrogen base, 0.25% ammonium sulfate, 1M sorbitol and SDC-Trp) plates until 30 colonies appear. Incubated for several days at 0C. SDC (Synthetic Dextrose Complete) is adenine, arginine, histidine, tryptophan and uracil 20 mg / l, leucine, lysine, tyrosine and isoleucine 30 mg / l, phenylalanine 50 mg / l, threonine 200 mg / l, valine 150 mg / l each.

To test for His ₆ -sbTNFα expression, some colonies were minimally containing YPD medium or MM + SDC-Trp (glucose 2%, yeast nitrogen base 0.17%, ammonium sulfate 0.25% and SDC-Trp. Medium) for several days. To check protein expression, whole cell extracts were ground with a Branson ultrasonic homogenizer with a period of 10 pulses for 30 seconds or with a bead beater using glass beads with a period of 8 pulses for 1 minute. After grinding, cell residues are collected by centrifugation and boiled in SDS-loading buffer solution for analysis by Western blot using SDS-PAGE and anti-polyhistidine mAb (Sigma). The supernatant His ₆ -sbTNFα was measured.

Expression of European sea bream sbTNFα in alcohol-utilizing yeast
Using TNF-ECOF and TNF-ECOR primers sbTNFα containing plasmid pET15b as the template in PCR with (see Figure 1) for amplification of the fragment containing the His ₆ -sbTNFα cloning 618pb to pPCICZA of His ₆ -sbTNFα. Both primers contain an EcoRI restriction site for cloning the PCR product within the same site of the plasmid pPCICZA (Invitrogen). Amplification is a sample containing 5 ng of template, 2.5 mM of MgCl ₂ , 50 μM of each dNTP, 0.2 mM of primers, 1X • High • Speec additive, 1 × buffer PLUS and 2 units of Eco • Taq • PLUS • DNA polymerase. Went in. Cycle reaction in Smart Cycler II (Cepheid) 1 cycle at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 30 seconds, 30 cycles at 55 ° C for 45 seconds and 30 cycles at 72 ° C for 2 minutes, Subsequently, 72 ° C. and 10 minutes were performed in the order of one cycle. PCR products were separated by electrophoresis in 0.8% agarose gel (Pronadisa) containing 0.5 μg / ml ethidium bromide and purified with QIAquick gel extraction kit (Qiagen). The purified fragment and 200 ng of pPICZA were digested with 10 units of EcoRI (manufactured by Fermentas) at 37 ° C. and then ligated at 22 ° C. using 5 units of T4 DNA ligase (manufactured by Fermentas). The ligation reaction product was transformed into E. coli TOP10F ′ competent cells, and plasmid-containing colonies were selected in an LB low salt plate having 25 μg / ml of Zeocin (Invitrogen). Recombinants were identified by colony PCR using 2X • PCR • Master • Mix (Fermentas) and PIC5 and PIC3 primers (see FIG. 1). The resulting His ₆ -sbTNFα-containing plasmid pPCICZA was purified with the QIAprep / Spin / Miniprep kit (Qiagen) and tested for insert orientation through digest release with EcoRI and digestion with Xhol. Moreover, the sequence of the plasmid was determined by ABI Prism 3130 gene analyzer (SACE, University of Murcia). In the recombinant plasmid, the star codon ATG of sbTNFα is present in the yeast consensus sequence AAAA ATG TCT (see FIG. 3) for foreign gene expression in yeast (see Romanos et al. 1992 paper). This consensus sequence is included in the primer TNF-ECOPP.

Electroporation and expression measurement of alcohol-utilizing yeast Before the electroporation of alcohol-utilizing yeast, the plasmid pPICZA containing His ₆ -sbTNFα was obtained for recombination and integration in the genomic DNA of alcohol-utilizing yeast. Linearized through digestion with Sacl or Pmel at 0C. A total amount of 5 μg of the linear plasmid was purified with the MiniElute Reaction Cleanup kit (Qiagen) and eluted with 10 μl of sterilized water. The alcohol-utilizing yeast strain used for electroporation was X-33. To make X-33 for electroporation, 100 ml cells of culture with OD ₆₀₀ = 1.5 were centrifuged at 1500 × g for 5 minutes at 4 ° C. After washing twice with sterile water, the cells were resuspended in 1M sorbitol, centrifuged as before, and finally resuspended in 100 ml of 1M sorbitol. In order to perform electroporation, 10 μl of DNA was mixed with 80 μl of cells and transferred to an ice-cooled 0.2 cm electroporation cuvette (manufactured by BioRad). After incubation for 5 minutes, a pulse was applied with a MicroPulser electroporator (BioRad) under the conditions of 25 μF, 1.5 kV, and 400Ω. Immediately thereafter, 1 ml of ice-cold 1M sorbitol solution was added to the cuvette and the resulting mixture was incubated at 30 ° C. for 1 hour. Cells were spread on YPDS plates with 100 μg / ml Zeocin (Invitrogen) and incubated at 30 ° C. for several days until colonies appeared. Several transformants were analyzed for the presence of the insert by PCR using PIC5 and PIC3 primers and 2X PCR master mix (Fermentas) and measured for His ₆ -sbTNFα expression. In specific colonies, genomic DNA was isolated using a genomic DNA purification kit (Fermentas), and His ₆ -sbTNFα-containing DNA fragments were amplified with EcoTaq / PULS / DNA polymerase (Ecogen), and ABI / Its sequence was determined by Prism 3130 gene analyzer (SACE, University of Murcia).

Recombinant alcohol-utilizing yeast strains with His ₆ -sbTNFα are grown in 25 ml glycerol-containing minimal medium at 30 ° C. and 250 rpm to generate biomass before methanol introduction until the culture reaches OD ₆₀₀ -6 It was. Cells were harvested by centrifugation at 3000 xg for 5 minutes and the pellet was resuspended in 200 ml of minimal medium containing 0.5% methanol until OD ₆₀₀ -1 was reached to induce expression. Cultures were incubated as described above and 0.5% methanol was added every 24 hours to maintain expression induction. At several time points samples were collected and analyzed for protein expression. To check protein expression, whole cell extracts were ground with a Branson ultrasonic homogenizer with a period of 10 pulses for 30 seconds or with a bead beater using glass beads with a period of 8 pulses for 1 minute. After grinding, cell residues are collected by centrifugation and boiled in SDS-loading buffer solution for analysis by Western blot using SDS-PAGE and anti-polyhistidine mAb (Sigma). The supernatant His ₆ -sbTNFα was measured.

Expression of seabass sbTNFα in Escherichia coli
Cloning of sbTNFα into pET15b
As a template in PCR for amplification of sbTNFα by FE4 and RE5 (see FIG. 1), cDNA obtained from seabass liver was used. Both primers have a BamHI restriction site for post cloning of the PCR product at the same site of plasmid pET15b (Novagen). Amplification was performed in a sample containing a cDNA template, each dNTP 50 μM, primer 0.2 mM, 1 × buffer PLUS containing MgCl ₂ and 1 unit of Eco Taq PULS DNA polymerase (manufactured by Ecogen). The cycle reaction is carried out in a smart cycler (Cepheid) for 1 cycle at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 45 seconds, 30 cycles at 55 ° C for 45 seconds and 30 cycles at 72 ° C for 90 seconds. A cycle of 72 ° C. for 10 minutes was performed in the order of one cycle. After separation by electrophoresis in 0.8% agarose gel (Pronadisa), the PCR product was purified with a QIAquick PCR purification kit (Qiagen). The purified fragment and the plasmid pET15b were digested with 10 units of BamHI at 37 ° C. for 2 hours, and the PCR purified product by the PCR purification kit was ligated at 22 ° C. with 1 unit of T4 DNA ligase (manufactured by Fermentas). . The ligation mixture was used against transformed E. coli DH5α competent cells (transformed recipient cells) and spread into ampicillin-containing LB plates. The plate was incubated at 37 ° C., and the recombinants were identified by colony PCR using 2 × PCR master mix (manufactured by fermentas) and primers T7F and T7R (see FIG. 1). Several generated colony plasmids were isolated with the QIAprep Spin Miniprep kit (Qiagen) and tested for insert release by BamHI digestion and insert orientation by Pvull digestion. The sequence of the recombinant plasmid was determined with an ABI Prism 3130 gene analyzer (SACE, manufactured by Murcia University).

E. coli transformation and expression measurement The transformation and expression measurement of the sea bass His ₆ -sbTNFα in E. coli were carried out in the same manner as described in Example 1.

Expression of seabass sbTNFα in alcohol-utilizing yeast
Cloning into His in ₆ -SbTNFarufa pPICZA Cloning sea bass to His ₆ -SbTNFarufa of pPICZA was performed in a similar procedure similar to that described in Example 3. The sequence of the resulting plasmid pPICZA containing the seabass His ₆ -sbTNFα was determined with an ABI Prism 3130 gene analyzer (SACE, manufactured by Murcia University). As described in Example 3, the star codon ATG of sbTNFα is present in the yeast consensus sequence AAAA ATG TCT (see FIG. 4) for foreign gene expression in yeast (see Romanos et al., 1992 paper). . This consensus sequence is included in the primer TNF-ECOPP.

Electroporation and expression measurement of alcohol-utilizing yeasts Electroporation was performed in the same manner as described in Example 3. However, plasmid linearization was performed with Pmel (manufactured by Fermentas). In the selected colonies, DNA is isolated with a genomic DNA purification kit (manufactured by Fermentas), a His ₆ -sbTNFα-containing DNA fragment is amplified with EcoTaq, PULS, DNA polymerase (manufactured by Ecogen), and ABIPrism3130 gene analyzer ( The sequence was determined by SACE (Murcia University).

  Expression measurement was also performed in the same manner as described in Example 3.

Expression of turbot sbTNFα in Escherichia coli
Cloning of His ₆ -sbTNFα into pET15b cDNA obtained from turbot liver was used as a template in the PCR method for sbTNFα amplification using primers TNFRO-BAMF and TNFRO-BAMR (see FIG. 1). Both primers have a BamHI restriction site for post cloning of the PCR product at the same site of plasmid pET15b (Novagen). Amplification was performed in a sample containing a cDNA template, each dNTP 50 μM, primer 0.2 mM, 1 × buffer PLUS containing MgCl ₂ and 1 unit of Eco Taq PULS DNA polymerase (manufactured by Ecogen). The cycle reaction is carried out in a smart cycler (Cepheid) for 1 cycle at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 45 seconds, 30 cycles at 55 ° C for 45 seconds and 30 cycles at 72 ° C for 90 seconds. A cycle of 72 ° C. for 10 minutes was performed in the order of one cycle. After separation by electrophoresis on a 0.8% agarose gel (Pronadisa), the PCR product was purified with a QIAquick PCR purification kit (Qiagen). The purified fragment and the plasmid pET15b were digested with 10 units of BamHI at 37 ° C. for 2 hours, and the PCR purified product by the PCR purification kit was ligated at 22 ° C. with 1 unit of T4 DNA ligase (manufactured by Fermentas). The ligation mixture was used against transformed E. coli DH5α competent cells (transformed recipient cells) and spread into ampicillin-containing LB plates. This plate was incubated at 37 ° C., and the recombinants were identified by colony PCR using 2 × PCR master mix (manufactured by fermentas). Several product colony plasmids were isolated and tested for insert release by BamHI digestion and insert orientation by Pvull digestion. The sequence of the selected plasmid was determined with an ABI Prism377 gene analyzer (CIB, manufactured by CSIC).

Transformation of E. coli and measurement of expression Transformation of E. coli and measurement of turbot His ₆ -sbTNFα expression were carried out in the same manner as described in Example 1.

Expression of turbot sbTNFα in alcohol-utilizing yeast
Cloning into pPICZA of His ₆ -sbTNFα cloning turbot into pPICZA of His ₆ -sbTNFα was performed in a similar procedure similar to that described in Example 3. The sequence of the resulting plasmid pPICZA containing turbot His ₆ -sbTNFα was determined with an ABI Prism 3130 gene analyzer (SACE, manufactured by Murcia University). As described in Example 3, the star codon ATG of sbTNFα is present in the yeast consensus sequence AAAA ATG TCT (see FIG. 5) for foreign gene expression in yeast (see Romanos et al., 1992 paper). . This consensus sequence is included in the primer TNF-ECOPP.

Electroporation and expression measurement of alcohol-utilizing yeasts Electroporation was performed in the same manner as described in Example 3. However, plasmid linearization was performed with Pmel (manufactured by Fermentas). In the selected colonies, DNA is isolated with a genomic DNA purification kit (manufactured by Fermentas), a His ₆ -sbTNFα-containing DNA fragment is amplified with EcoTaq, PULS, DNA polymerase (manufactured by Ecogen), and ABIPrism3130 gene analyzer ( The sequence was determined by SACE (Murcia University).

  Expression measurement was also performed in the same manner as described in Example 3.

Purification of sbTNFα by chromatographic method His ₆ -sbTNFα expressed in Escherichia coli, budding yeast or alcohol-utilizing yeast was purified by affinity chromatography using AKTA · Explorer · FPLC (manufactured by GE Healthcare). Purification was performed using a HisTrap • FF column (manufactured by GE Healthcare) by a fixed metal ion affinity chromatography method using Ni ²⁺ (purification method of histidine-labeled protein). Absorption at 280 nm was measured by UNICORN 5.10. A 1 ml column was equilibrated with at least 5 column volumes of binding buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 5 mM imidazole, pH 7.4) at a flow rate of 1 ml / min. Next, the growth culture sample was pulverized with an ultrasonic wave or a bead beater, and further centrifuged to remove the pellet to obtain a clear supernatant. As described in Example 2, immediately after this, 2 mL of this supernatant was loaded into the column. The column was washed with binding buffer until the absorption reached a stable baseline (10-15 column volumes). Finally, a 20-25 column volume linear gradient was applied to increase the amount of elution buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 5 mM imidazole, pH 7.4) to elute the protein bound to the column. . Using an automatic fraction collector, 1 ml fractions of the eluate were collected. Protein-containing fractions were tested for the presence of His ₆ -sbTNFα by SDS-PAGE and immunoblotting using anti-polyhistidine mAb (Sigma). The selected fractions were combined, and the amount of His ₆ -sbTNFα was measured by Bradfold (manufactured by Sigma).

If necessary, the His ₆ tag (label) was removed using thrombin-agarose (Recom-T, Sigma).

Detection of sbTNFα by SDS-PAGE and immunoblotting Samples with His ₆ -sbTNFα were analyzed by SDS-polyacrylimide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide / bisacrylamide. The gel was stained with coomassie® 0.1% for 30 minutes to distinguish a 21 kDa band corresponding to His ₆ -sbTNFα.

To detect His ₆ -sbTNFα by performing immunoblotting after SDS-PAGE as described above, the gel was placed in TBS (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20) at 100 V for 1 hour. To a nitrocellulose membrane (manufactured by Sigma). The membrane was incubated with TTBS with 5% BSA (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween20) to block proteins at 4 ° C. After washing the membrane with TTBS, it was incubated with TTBS + 5% BSA + 1: 1000 anti-polyhistidine mAb (Sigma) for 2 hours. The membrane was then washed again with TTBS and TBS before staining with ECL (Amersham Biosciences). A 21 kDa single band corresponding to His ₆ -sbTNFα appeared (see FIG. 6).

Culture of yeast by fermentation Culture of the transformed yeast by fermentation was performed with a bioreactor system (Applicon).

As an example, an alcohol-utilizing yeast strain was cultured in a flask having a YPGly medium until the OD ₆₀₀ of the culture reached 30 or more. Growth cultures were then inoculated in a bioreactor system containing MMG (glycerol-containing minimal medium). The culture conditions were as follows. Temperature 30 ° C., pH 5.00 (adjusted by adding NH ₃ ), stirring speed 500-1250 rpm, oxygen concentration 30%, cascade 30% with stirring. These parameters were measured by BioXpert · V2. Fermentation was carried out in a batch process until the OD _{600 of} the culture reached 50-80. Immediately after the OD _{600 of the} culture reached 50-80, the fermentation was changed to a semi-batch culture and feeding with glycerol-containing feed continued for 10 hours (OD ₆₀₀ of the culture reached 260-280). Finally, feeding with a methanol-containing feed was continued for 40 hours. Eventually, the OD ₆₀₀ of the culture reached 380-420.

Expression analysis of recombinant TNF His ₆ -sbTNFα was performed by immunoblotting (see Example 9).

After the microencapsulated fermentation of the recombinant yeast has reached the desired biomass and the optimal expression of the recombinant TNF · His ₆ -sbTNFα has been obtained, the culture fluid is aseptically collected, and 20% malodextrin and 10% It was formulated using a protective polymer Kollicoat (registered trademark) MAE100P (manufactured by BASF).

  The formulated suspension was then ejected from a spray dryer (Buchi). The inlet temperature was controlled at 120 ° C and the outlet temperature at 85 ° C.

The final microcapsules of recombinant yeast were analyzed and the absence of cell viability and the presence of TNF His ₆ -sbTNFα was determined by immunoblotting (see Example 10).

Enrichment of Artemia nauplii larvae (Artemia nauplii) with microencapsulated recombinant yeast Artemia nauplii larvae were hatched in a flask containing 500 ml of sterilized complete seawater. Seawater in the flask was aerated by an air hose connected to an aquarium air pump and maintained at 28 ° C. in a reciprocating shaking water bath. Nauplius larvae were collected in the enrichment system 24 hours after hatching. This system consists of a plurality of flasks with a capacity of 100 ml, each flask is filled with fresh seawater, each flask is all placed in the same water bath and aerated separately. For all experiments, hatchability and the number of nauplius larvae were estimated by taking 0.5 ml samples of seawater.

  The optimum production time of Artemia naprius larvae enriched with microencapsulated recombinant yeast was evaluated by staining the yeast with DTAF. The stained yeast was then used to feed Artemia naprius larvae. Artemia naprius larvae were killed at 0, 30, 45, 60 and 120 minutes after feeding. Enriched Artemia naprius larvae were observed by fluorescence microscopy.

  The results are shown in FIG. As shown, after the start of feeding, initially an increase in the amount of microencapsulated recombinant yeast is observed in the oral region, after which the entire intestine is filled with microencapsulated recombinant yeast, Cells were not observed.

  The optimum production time for enriched Artemia naprius larvae was 60 minutes.

Expression and secretion of European sea bream sbTNF.ALPHA. In alcohol-utilizing yeast
For amplification of the His ₆ -sbTNFα containing fragment cloning 615pb to pPICZαA of His ₆ -sbTNFα, using sbTNFα containing plasmid pET15b as the template in the PCR method by TNF-ECOF and TNF-ECOR (see FIG. 1). Both primers contain an EcoRI restriction site for cloning the PCR product within the same site of plasmid pPICZαA. For amplification, 5 ng of template, 2.5 mM of MgCl ₂ , 50 μM of each dNTP, 0.2 mM of primer, 1X High Speec additive, 1X buffer PLUS and Eco • Taq • PLUS • DNA polymerase (manufactured by Ecogen) In a sample containing 2 units. Cycle reaction in Smart Cycler II (Cepheid) 1 cycle at 95 ° C for 5 minutes, 30 cycles at 95 ° C for 30 seconds, 30 cycles at 55 ° C for 45 seconds and 30 cycles at 72 ° C for 2 minutes, Subsequently, 72 ° C. and 10 minutes were performed in the order of one cycle. PCR products were separated by electrophoresis in 0.8% agarose gel (Pronadisa) containing 0.5 μg / ml ethidium bromide and purified with QIAquick gel extraction kit (Qiagen). The purified fragment and 200 ng of pPICZαA were digested with 10 units of EcoRI (manufactured by Fermentas) at 37 ° C. and then ligated at 22 ° C. with 5 units of T4 DNA ligase (manufactured by Fermentas). The ligation reaction product was transformed into E. coli TOP10F ′ competent cells, and plasmid-containing colonies were selected in an LB plate having 25 μg / ml of Zeocin (Invitrogen). Recombinants were identified by colony PCR using 2X • PCR • Master • Mix (manufactured by Fermentas) and PICα and PIC3 primers. The resulting His ₆ -sbTNFα-containing plasmid pPICZαA was purified with a QIAprep / Spin / Miniprep kit (manufactured by Qiagen), and tested for insert release after digestion with EcoRI and insert orientation with digested Smal and Ncol. Moreover, the sequence of the plasmid was determined by ABI Prism 3130 gene analyzer (SACE, University of Murcia).

Electroporation and expression measurement of alcohol-utilizing yeast. Before electroporation of alcohol-utilizing yeast, the His ₆ -sbTNFα-containing plasmid pPICZαA was used for recombination and integration in the genomic DNA of alcohol-utilizing yeast. Linearized through digestion with Sacl or Pmel at 0C. A total amount of 5 μg of the linear plasmid was purified with the MiniElute Reaction Cleanup kit (Qiagen) and eluted with 10 μl of sterilized water. The alcohol-utilizing yeast strain used for electroporation was X-33. To make X-33 for electroporation, 100 ml cells of culture with OD ₆₀₀ = 1.5 were centrifuged at 1500 × g for 5 minutes at 4 ° C. After washing twice with sterile water, the cells were resuspended in 1M sorbitol, centrifuged as before, and finally resuspended in 100 ml of 1M sorbitol. In order to perform electroporation, 10 μl of DNA was mixed with 80 μl of cells and transferred to an ice-cooled 0.2 cm electroporation cuvette (manufactured by BioRad). After incubation for 5 minutes, a pulse was applied with a MicroPulser electroporator (BioRad) under the conditions of 25 μF, 1.5 kV, and 400Ω. Immediately thereafter, 1 ml of ice-cold 1M sorbitol solution was added to the cuvette and the resulting mixture was incubated at 30 ° C. for 1 hour. Cells were spread on YPDS plates with 100 μg / ml Zeocin (Invitrogen) and incubated at 30 ° C. for several days until colonies appeared. Several transformants were analyzed for the presence of the insert by PCR using PIC5 and PIC3 primers and 2X PCR master mix (Fermentas) and measured for His ₆ -sbTNFα expression. In specific colonies, genomic DNA was isolated using a genomic DNA purification kit (Fermentas), and His ₆ -sbTNFα-containing DNA fragments were amplified with EcoTaq / PULS / DNA polymerase (Ecogen), and ABI / Its sequence was determined by Prism 3130 gene analyzer (SACE, University of Murcia).

Recombinant alcohol-utilizing yeast strains with His ₆ -sbTNFα are grown in 25 ml glycerol-containing minimal medium at 30 ° C. and 250 rpm to generate biomass before methanol introduction until the culture reaches OD ₆₀₀ -6 It was. Cells were harvested by centrifugation at 3000 xg for 5 minutes and the pellet was resuspended in 200 ml of minimal medium containing 0.5% methanol until OD ₆₀₀ -1 was reached to induce expression. Cultures were incubated as described above and 0.5% methanol was added every 24 hours to maintain expression induction. At several time points samples were collected and analyzed for protein expression. To check protein expression, the supernatant His ₆ − was boiled in SDS-loading buffer for analysis by SDS-PAGE and anti-polyhistidine mAb (Sigma) by Western blot. sbTNFα was measured.

Samples of healthy European sea bream (Sparus aurata L.) ( Teleostei, Periformidae, Thaiaceae) weighing 120 g were micro-encapsulated recombinant yeast were raised in a 2 m ³ swimming seawater tank. 6 ppm of oxygen was dissolved in seawater at a flow rate of 20% tank capacity / hour. The aquarium was maintained at room temperature and day length, and was fed twice a day with a commercial pellet feed (Trouvit, located in Burgos, Spain). The test group was fed every 2 days with a basal feed supplemented with 0.1, 1 or 10% of control yeast or mature (active) sbTNFα overexpressing microencapsulated yeast. At all sampling times (2, 4, 6, and 10 days after treatment), the sample fish were weighed, the head kidney and intestine were collected, examined by light microscopy, and gene expression was examined. . The test methods described here follow the guidelines of the European Union Council (86/609 / EU) on the use of laboratory animals and the bioethics committee of the University of Murcia, Spain. Respiratory burst activity was measured as luminol-dependent chemiluminescence (see Sepulcre et al. 2007) produced by head kidney leucosite suspension after different stimulation times. All tests were performed using 5 fish and triplicate samples. The same test was repeated twice to ensure the validity of the results. The SPSS 13.0.1 statistical software package was used for all statistical analyses. Data was analyzed by square deviation (ANOVA) and Tukey multiple range tests to determine differences between test groups.

  Fish appetite, growth rate, swimming behavior and external morphology were not affected by treatment. In addition, no fish died during the test. Furthermore, no histopathological lesions that were not infiltrated with phagocytic cells (ie, eosinophilic granules or macrophages) were observed in the intestinal tract of fish fed with yeast containing sbTNFα for 10 days. Administration of yeast alone had little effect on fish immune status as measured as respiratory burst of head kidney leucosite. In marked contrast, fish feed containing 0.1 and 1% of over-expressed sbTNFα-containing microencapsulated yeast shows a respiratory burst that increases with dose after 4 days of treatment. (See FIG. 8). Surprisingly, with the exception of TLR9, yeast administration had little effect on the mRNA levels of several inflammatory genes in the head kidney and intestine. TLR9 expression was significantly upregulated (see FIG. 9).

  The above results show that administration of the immunostimulant for 10 days has no side effects on fish. Because respiratory burst is widely used as an indicator of fish immune status (Mulero et al., 1988 paper, Garcia-Castillo et al. 2004 paper, Chaves-Pozo et al. 2005 paper, and Sepulcre et al. 2007 This immune response activation in fish feed with TNFα-containing yeast is very effective for the immunostimulatory agent of the present invention, resulting in abiotic stress (ie, fish manipulation) And protects fish against biological stress (ie, stress caused by pathogens). Furthermore, the upregulation (ie, overexpression) of TLR9 expression in the intestinal tract by administration of the immunostimulatory agent of the present invention also suggests that the fish intestinal tract is well protected against viral and bacterial infections. Yes. This is because the receptor is a major component in the recognition of these pathogens (see Ishii and Akira 2006 paper). Overall, all the above results also indicate that recombinant sbTNFα is an excellent adjuvant for oral vaccines due to the systematic activation of innate immune cells and increased expression of TLR9.

The above description relates to one embodiment of the present invention, and those skilled in the art can consider various modifications of the present invention, all of which are included in the technical scope of the present invention. The
With respect to the term “or”, for example, “A or B” includes selecting “both A and B” as well as “A only” and “B only”. Unless stated otherwise, the number of devices or means may be singular or plural.

Claims (8)

(a) selected cytokine coding That cDNA tissue to decrypt the steps of: selecting from the fish, and isolated,
(b) cloning the cDNA into at least an expression vector for cytokine expression in at least a yeast host microorganism;
(c) culturing at least a host cell of a yeast host microorganism to obtain a recombinant cytokine or a culture of at least a yeast host microorganism containing the recombinant cytokine;
(d) a step of microencapsulating the recombinant cytokine or at least a yeast host microorganism containing the recombinant cytokine to obtain a microencapsulated recombinant cytokine or a microencapsulated yeast host microorganism containing the recombinant cytokine. A method for producing an orally administrable immunostimulant characterized by the above. The production method according to claim 1, wherein the at least yeast host microorganism is selected from methanol-utilizing yeast (Pichia pastoris), budding yeast (Saccharomyces cereviciae), and Kluyveromyces lactis. The (d) microencapsulation step is performed by spray-drying in the presence of an enteric protective polymer selected from cellulose acetate phthalate, hydroxypropylcellulose phthalate, carboxymethylcellulose, methacrylic acid copolymer, and chitosan. the method according to any one of claims 1-2, characterized in that dividing. A method for using an orally administrable immunostimulant produced by the production method according to any one of claims 1 to 3 for immune stimulation of fish aquacultured, wherein the fish is a European sea bream (Sparus aurata) , European seabass (Dicentrarchus labrax), rainbow trout (Oncorhynchus mykiss), shrimp (Psetta maxima), striped sea bream (Dentex dentex), Sharpsnout sea bream (Diplodus puntazzo), spanish sea bream (Pagellus bogaraveo), corbina (regius) A method of using an orally administrable immunostimulant characterized by being selected from (Anguilla anguilla). A method for using an orally administrable immunostimulant produced by the production method according to any one of claims 1 to 3 for immune stimulation of fish cultured in aquaculture, wherein the recombinant cytokine is immunostimulated. Use of an orally administrable immunostimulant characterized in that it is derived from the same fish species to be administered. The method according to any one of claims 4 to 5, wherein the immunostimulation is performed by administering the immunostimulant together with feed during feeding. The immune stimulation is
(a) administering an orally administrable immunostimulant to a multicellular organism selected from Artemia (Artemia sp.) and rotifer (phylum Rotifera);
(b) obtaining an enriched multicellular organism;
(c) subsequently feeding the enriched multicellular organism to a fish species;
The method according to any one of claims 4 to 6, wherein the method is used. A method of using an orally administrable immunostimulant produced by the production method according to any one of claims 1 to 3 as a vaccine adjuvant in aquaculture.